Dr Cartwright is a Professor of Medicine and Gastroenterology with tenure at Stanford University. She trained at the University of California San Diego and the Salk Institute for Biological Studies before joining the Stanford Faculty in 1989. Her research in cancer biology focuses on understanding how normal intestinal cells regulate their growth and how loss of that regulation results in malignant transformation. She is an author on numerous scientific publications. Her research has been supported by grants from the National Institutes of Health (for 30 years), the American Cancer Society, the Broad Medical Research Foundation and the Crohn’s and Colitis Foundation of America. She has served on NIH and American Cancer Society Study Sections and on the Editorial Board of the Gastrointestinal and Liver Physiology section of the American Journal of Physiology. In 1995 she was inducted into the American Society for Clinical Investigation. In 2008 she received an Outstanding Woman in Science Award from the American Gastroenterological Association (AGA) for her contributions to GI research. She has been an invited speaker, organizer and chairperson for numerous national AGA symposia on the molecular biology of gastrointestinal cancers.
Dr Cartwright’s clinical interests focus on caring for those with inflammatory bowel diseases. She has cared for nearly 5,000 IBD patients over the past 3 decades. She served as Director of the Program for Inflammatory Bowel Diseases at Stanford University (1989-2007). She also served on the Editorial Board of the Inflammatory Bowel Diseases Journal and on the National Scientific Advisory Committee (Clinical Affairs/Professional Education) of the Crohn’s and Colitis Foundation of America. In 2000 she received the Premier Physician Award from the Crohn’s & Colitis Foundation of America Greater Bay Area Chapter. She has been an invited speaker, organizer and chairperson for numerous IBD symposia in northern California. In 2020 she was nominated for the Master Clinician Award in the Department of Medicine, Stanford University School of Medicine.
- Inflammatory Bowel Diseases
Director, Program for Inflammatory Bowel Diseases, Stanford University School of Medicine (1989 - 2007)
Honors & Awards
Elected Member, American Society for Clinical Investigation (1995 - present)
Outstanding AGA Woman in Science Award, American Gastroenterological Association (2008)
Premier Physician Award, Crohn's & Colitis Foundation of America - Greater Bay Area Chapter (2000)
Honoree, Inflammatory Bowel Diseases Symposium, Stanford University School of Medicine (2015)
Health Care Honorary Award: Patient Satisfaction Scores > 90th %ile of physicians ranked nationally, Stanford University School of Medicine (2017 - present)
Nominee, Master Clinician Award, Department of Medicine, Stanford University School of Medicine (2020)
Boards, Advisory Committees, Professional Organizations
Member, Tumor Biochemistry/Endocrinology Study Section, American Cancer Society (1995 - 1999)
Member (Ad Hoc), General Medicine A-2 Study Section, National Institutes of Health (1998 - 1998)
Member, Gastrointestinal Cell / Molecular Biology Study Section, National Institutes of Health (2007 - 2009)
Editorial Board, American Journal of Physiology: Gastrointestinal and Liver Physiology (1997 - 2000)
Editorial Board, Inflammatory Bowel Diseases Journal (1994 - 2006)
Member, National Scientific Advisory Committee, Clinical Affairs/Professional Education, Crohn's and Colitis Foundation of America (1994 - 1997)
Co-Director, Medical Advisory Committee, Crohn's & Colitis Foundation of America, Greater Bay Area Chapter (1991 - 1997)
Member, Board of Trustees / Executive Committee, Crohn's & Colitis Foundation of America, Greater Bay Area Chapter (1991 - 1999)
Medical Advisory Board, Crohn's & Colitis Foundation of America, Greater Bay Area Chapter (1999 - 2001)
Fellowship: UCSD Medical Center Gastroenterology Fellowship (1984) CA
Residency: UCSD Internal Medicine Residency (1981) CA
Internship: UCSD Internal Medicine Residency (1979) CA
BS, Stanford University, Biology (1973)
MD, University of Utah, Medicine (1978)
Research Associate, The Salk Institute, Cancer Biology (1989)
Board Certification: American Board of Internal Medicine, Internal Medicine (1981)
Board Certification: American Board of Internal Medicine, Gastroenterology (1987)
Community and International Work
Education on Inflammatory Bowel Diseases, San Francisco Bay Area
Crohn's and Colitis Foundation of America
people with Inflammatory Bowel Diseases
Opportunities for Student Involvement
Inflammatory Bowel Diseases
Clinical Care and Education
Crohn's and Colitis Foundation of America
Opportunities for Student Involvement
Current Research and Scholarly Interests
Research in my laboratory focuses on molecular mechanisms of intestinal cell growth control. A primary focus is on function and regulation of the Src family of tyrosine kinases in normal cells, and their deregulation in cancer cells. Molecular, cellular and physiologic approaches are used to explore basic questions about growth regulation. Areas of active investigation include studies of Src function in cell cycle progression, proliferation, differentiation, adhesion, survival and malignant transformation; discovery of endogenous inhibitors of Src kinases; analysis of inhibitor function in cell growth control and apoptosis; and exploration of new drug therapy for colon cancer. Our recent discovery of a Src inhibitor, RACK1, which works both to inhibit growth (by suppressing Src activity at G1 and mitotic checkpoints) and to induce death of colon cells, could be exploited for development of new and more powerful and selective strategies for treatment of human colon cancer.
Independent Studies (7)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
Graduate and Fellowship Programs
Gastroenterology & Hepatology (Fellowship Program)
Rack1 maintains intestinal homeostasis by protecting the integrity of the epithelial barrier
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
2018; 314 (2): G263–G274
Previously, we generated mouse models of Rack1 deficiency to identify key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. However, other than low body weight, we did not detect an overt phenotype in mice constitutively deleted of Rack1 in intestinal epithelia ( vil-Cre: Rack1fl/fl mice), presumably because Rack1 was deleted in <10% of the total surface area of the epithelia. To assess the effect of Rack1 loss throughout the entire intestinal epithelia, we generated another mouse model of Rack1 deficiency, vil-Cre-ERT2: Rack1fl/fl. Within 5-10 days of the initial tamoxifen treatment, the mice lost over 20% of their body weight, developed severe diarrhea that for some was bloody, became critically ill, and died, if not euthanized. Necropsies revealed mildly distended, fluid-, gas-, and sometimes blood-filled loops of small and large bowel, inguinal lymphadenopathy, and thrombocytosis. Rack1 was deleted in nearly 100% of the epithelia in both the small intestine and colon when assessed by immunofluorescent or immunoblot analyses. Rack1 expression in other tissues and organs was not different than in control mice, indicating tissue specificity of the recombination. Histopathology revealed a patchy, erosive, hemorrhagic, inflammatory enterocolitis with denuded, sloughed off surface epithelium, and crypt hyperplasia. These results suggest a protective function for Rack1 in maintaining the integrity of intestinal epithelia and for survival. NEW & NOTEWORTHY Our findings reveal a novel function for Rack1 in maintaining intestinal homeostasis by protecting the epithelial barrier. Rack1 loss results in a patchy, erosive, hemorrhagic, inflammatory enterocolitis, which resembles that of inflammatory bowel diseases (IBD) in humans. Understanding mechanisms that protect barrier function in normal intestine and how loss of that protection contributes to the pathogenesis of IBD could lead to improved therapies for these and other erosive diseases of the gastrointestinal tract.
View details for DOI 10.1152/ajpgi.00241.2017
View details for Web of Science ID 000425923800012
View details for PubMedID 29025732
View details for PubMedCentralID PMC5866421
Rack1 function in intestinal epithelia: regulating crypt cell proliferation and regeneration and promoting differentiation and apoptosis
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
2018; 314 (1): G1–G13
Previously, we showed that receptor for activated C kinase 1 (Rack1) regulates growth of colon cells in vitro, partly by suppressing Src kinase activity at key cell cycle checkpoints, in apoptotic and cell survival pathways and at cell-cell adhesions. Here, we generated mouse models of Rack1 deficiency to assess Rack1's function in intestinal epithelia in vivo. Intestinal Rack1 deficiency resulted in proliferation of crypt cells, diminished differentiation of crypt cells into enterocyte, goblet, and enteroendocrine cell lineages, and expansion of Paneth cell populations. Following radiation injury, the morphology of Rack1-deleted small bowel was strikingly abnormal with development of large polypoid structures that contained many partly formed villi, numerous back-to-back elongated and regenerating crypts, and high-grade dysplasia in surface epithelia. These abnormalities were not observed in Rack1-expressing areas of intestine or in control mice. Following irradiation, apoptosis of enterocytes was strikingly reduced in Rack1-deleted epithelia. These novel findings reveal key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. NEW & NOTEWORTHY Our findings reveal novel functions for receptor for activated C kinase 1 (Rack1) in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia.
View details for DOI 10.1152/ajpgi.00240.2017
View details for Web of Science ID 000423762000001
View details for PubMedID 28935684
View details for PubMedCentralID PMC5866376
- Pulmonary Crohn's Disease DIGESTIVE DISEASES AND SCIENCES 2017; 62 (1): 64-67
Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity
Cell Stem Cell
2017; 21 (1): 78 - 90.e6
Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ISCs, the most well-defined ISC pool, but Bmi1-GFP+cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.
View details for DOI 10.1016/j.stem.2017.06.014
View details for PubMedCentralID PMC5642297
Rack1 promotes epithelial cell-cell adhesion by regulating E-cadherin endocytosis
2012; 31 (3): 376-389
E-cadherin and its cytoplasmic partners, catenins, mediate epithelial cell-cell adhesion. Disruption of this adhesion allows cancer cells to invade and metastasize. Aberrant activation of the Src tyrosine kinase disrupts cell-cell contacts through an E-cadherin/catenin-dependent mechanism. Previously we showed that Rack1 regulates the growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and in the intrinsic apoptotic and Akt cell survival pathways. Here we show that Rack1, partly by inhibiting Src, promotes cell-cell adhesion and reduces the invasive potential of colon cancer cells. Rack1 stabilizes E-cadherin and catenins at cell-cell contacts by inhibiting the Src phosphorylation of E-cadherin, the ubiquitination of E-cadherin by the E3 ligase Hakai and the endocytosis of E-cadherin. Upon depletion and restoration of extracellular calcium, Rack1 facilitates the re-assembly of E-cadherin-containing cell-cell contacts. Rack1 also blocks HGF-induced endocytosis of E-cadherin, disruption of cell-cell contacts and cell scatter. Our results uncover a novel function of Rack1 in maintaining the junctional homeostasis of intestinal epithelial cells by regulation of the Src- and growth factor-induced endocytosis of E-cadherin.
View details for DOI 10.1038/onc.2011.242
View details for Web of Science ID 000299542100010
View details for PubMedID 21685945
- Relapse of Intestinal and Hepatic Amebiasis After Treatment DIGESTIVE DISEASES AND SCIENCES 2011; 56 (3): 677-680
Characterization of In Vivo Keratin 19 Phosphorylation on Tyrosine-391
2010; 5 (10)
Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Although K19 is known to be phosphorylated on tyrosine residue(s), conclusive site-specific characterization of these residue(s) and identification potential kinases that may be involved has not been reported.In this study, biochemical, molecular and immunological approaches were undertaken in order to identify and characterize K19 tyrosine phosphorylation. Upon treatment with pervanadate, a tyrosine phosphatase inhibitor, human K19 (hK19) was phosphorylated on tyrosine 391, located in the 'tail' domain of the protein. K19 Y391 phosphorylation was confirmed using site-directed mutagenesis and cell transfection coupled with the generation of a K19 phospho (p)-Y391-specific rabbit antibody. The antibody also recognized mouse phospho-K19 (K19 pY394). This tyrosine residue is not phosphorylated under basal conditions, but becomes phosphorylated in the presence of Src kinase in vitro and in cells expressing constitutively-active Src. Pervanadate treatment in vivo resulted in phosphorylation of K19 Y394 and Y391 in colonic epithelial cells of non-transgenic mice and hK19-overexpressing mice, respectively.Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein. The lack of detection of K19 pY391 in the absence of tyrosine phosphatase inhibition suggests that its phosphorylation is highly dynamic.
View details for DOI 10.1371/journal.pone.0013538
View details for Web of Science ID 000283422100006
View details for PubMedID 21049038
View details for PubMedCentralID PMC2963603
- Distal Extrahepatic Cholangiocarcinoma Presenting as Cholangitis DIGESTIVE DISEASES AND SCIENCES 2010; 55 (7): 1852-1855
A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways
2009; 28 (50): 4421-4433
Earlier we showed that RACK1 regulates growth of human colon cells by suppressing Src activity at G(1) and mitotic checkpoints. Here, we show that RACK1 also induces apoptosis of the cells, partly by inhibiting Src. In the intrinsic pathway, RACK1 inhibits expression of anti-apoptotic Bcl-2 and Bcl-X(L), induces expression of pro-apoptotic Bim, targets Bim and Bax to the mitochondria, induces oligomerization of Bax (which requires Bim and inhibition of Src), depolarizes mitochondria membranes, releases cytochrome c, and activates caspases-9 and -3 and death substrates. Bax and Bim are required for RACK1-mediated mitochondrial cell death. RACK1-induced oligomerization of Bax is required for staurosporine-mediated cell death. RACK1 also induces apoptosis by blocking Src activation of the Akt cell survival pathway. This leads to activation of the transcription factor FOXO3, a potent inducer of apoptosis and G(1) arrest. Collectively, our results show that RACK1, partly by inhibiting Src, promotes mitochondrial cell death and blocks Akt-mediated cell survival. Thus, RACK1 inhibits growth and induces death of colon cells. Exploitation of these dual functions could lead to novel colon cancer therapies that mimic RACK1 function.
View details for DOI 10.1038/onc.2009.293
View details for Web of Science ID 000272876500002
View details for PubMedID 19767770
- Crohn's Colitis Complicated by Cytomegalovirus Infection DIGESTIVE DISEASES AND SCIENCES 2009; 54 (9): 1864-1867
RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints
2007; 26 (20): 2914-2924
Previously, we showed that Src tyrosine kinases are activated early in the development of human colon cancer and are suppressed as intestinal cells differentiate. We identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src. Here we show (by overexpressing RACK1, depleting Src or RACK1 and utilizing cell-permeable peptides that perturb RACK1's interaction with Src) that RACK1 regulates growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and consequently delaying cell cycle progression. Activated Src rescues RACK1-inhibited growth of HT-29 cells. Conversely, inhibiting Src abolishes growth promoted by RACK1 depletion in normal cells. Two potential mechanisms whereby RACK1 regulates mitotic exit are identified: suppression of Src-mediated Sam68 phosphorylation and maintenance of the cyclin-dependent kinase (CDK) 1-cyclin B complex in an active state. Our results reveal novel mechanisms of cell cycle control in G(1) and mitosis of colon cells. The significance of this work lies in the discovery of a mechanism by which the growth of colon cancer cells can be slowed, by RACK1 suppression of an oncogenic kinase at critical cell cycle checkpoints. Small molecules that mimic RACK1 function may provide a powerful new approach to the treatment of colon cancer.
View details for DOI 10.1038/sj.onc.1210091
View details for Web of Science ID 000246210800011
View details for PubMedID 17072338
Peptide modulators of Src activity in G(1) regulate entry into S phase and proliferation of NIH 3T3 cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2007; 352 (2): 423-430
Cascades of kinases and phosphatases are regulated by selective protein-protein interactions that are essential for signal transduction. Peptide modulators of these interactions have been used to dissect the function of individual components of the signaling cascade, without relying on either the over- or underexpression of proteins. Previously, we identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src tyrosine kinases. Here, we utilized cell-permeable peptides that selectively disrupt or enhance the interaction of RACK1 and Src to further examine the function of RACK1. Our results provide direct physiologic evidence that RACK1 regulates growth of NIH3T3 cells by suppressing the activity of Src and other cell cycle regulators in G1, and delaying entry into S phase. They also demonstrate the potential for using peptide modulators of Src activity as a tool for regulating cell growth, and for designing new strategies for cancer therapy that target specific protein-protein interactions.
View details for DOI 10.1016/j.bbrc.2006.11.034
View details for PubMedID 17118337
Human colon cancer cells lack mutations in RACK1, a suppressor of oncogenic Src tyrosine kinases
71st Annual Scientific Meeting of the American-College-of-Gasroenterology
NATURE PUBLISHING GROUP. 2006: S198–S198
View details for Web of Science ID 000240656100460
RACK1 regulates G(1)/S progression by suppressing Src kinase activity
MOLECULAR AND CELLULAR BIOLOGY
2004; 24 (15): 6788-6798
Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.
View details for DOI 10.1128/MCB.24.15.6788-6798.2004
View details for PubMedID 15254245
RACK1 regulates Src-mediated Sam68 and p190RhoGAP signaling
2004; 23 (33): 5682-5686
RACK1 is the founding member of a family of receptors for activated C kinase collectively called RACKs. Upon activation of PKC, RACK1 co-localizes with the Src tyrosine kinase at the plasma membrane and functions as a substrate, binding partner and inhibitor of Src (as measured in vitro), and a growth inhibitor in NIH 3T3 cells. To further analyze the function of RACK1 in Src and PKC signaling, we utilized cell-permeable peptides that modulate the interaction of RACK1 and betaIIPKC, thereby affecting betaIIPKC translocation and function. We found that the association of betaIIPKC and RACK1 is necessary for Src phosphorylation of RACK1. Src activity is required for tyrosine phosphorylation of RACK1, and for RACK1 binding to Src, but not to betaIIPKC. Endogenous Src kinase activity, as measured by phosphorylation of Sam68 (a mitotic-specific Src substrate involved in cell cycle regulation and RNA splicing) or p190RhoGAP (a Src substrate and GTPase-activating protein involved in actin reorganization), increases with disruption of the Src-RACK1 complex, and decreases with enhanced complex formation. RACK1 inhibits Src-mediated p190RhoGAP signaling and actin cytoskeleton rearrangement. Thus, RACK1 functions as an endogenous inhibitor of the Src kinase in diverse signaling pathways that regulate distinct cellular functions. Our results demonstrate the potential for using peptide modulators of Src activity as a tool for uncovering the function of Src in cells.
View details for DOI 10.1038/sj.onc.1207735
View details for PubMedID 15184885
RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells
2004; 567 (2-3): 321-326
Cancer cells are capable of serum- and anchorage-independent growth, and focus formation on monolayers of normal cells. Previously, we showed that RACK1 inhibits c-Src kinase activity and NIH3T3 cell growth. Here, we show that RACK1 partially inhibits v-Src kinase activity, and the serum- and anchorage-independent growth of v-Src transformed cells, but has no effect on focus formation. RACK1-overexpressing v-Src cells show disassembly of podosomes, which are actin-rich structures that are distinctive to fully transformed cells. Together, our results demonstrate that RACK1 overexpression in v-Src cells partially reverses the transformed phenotype of the cells. Our results identify an endogenous inhibitor of the oncogenic Src tyrosine kinase and of cell transformation.
View details for DOI 10.1016/j.febslet.2004.03.125
View details for Web of Science ID 000221890000031
View details for PubMedID 15178345
Cytomegalovirus infection in steroid-refractory ulcerative colitis - A case-control study
AMERICAN JOURNAL OF SURGICAL PATHOLOGY
2004; 28 (3): 365-373
Cytomegalovirus (CMV) infection is reported to be a cause of steroid-refractory ulcerative colitis (UC), but the strength of this association has not been tested in a case control study. Controlled studies have also not been performed to determine the sensitivity of available immunohistochemical techniques to detect CMV in this setting. The pathology database at Stanford Hospital was searched for UC patients with a diagnosis of "severe colitis" between the years 1992 and 2002 and medical records were reviewed. Forty patients were identified with refractory UC, defined as poor response to highdose systemic steroids for >2 weeks. Another group of 40 patients with severe, but nonrefractory, UC was case-matched for age and year of biopsy. A series of 40 patients who underwent colectomy for reasons other than inflammatory bowel disease with representative sections of "normal" colon were selected as noncolitis controls. CMV inclusions were detected on hematoxylin and eosin (H&E) in 2 of 40 patients with refractory UC, but not in other patients. Immunohistochemistry (IHC) detected CMV in 10 of 40 (25%) patients with refractory UC and 1 of 40 (2.5%) patients with nonrefractory UC (P = 0.007). The CMV-positive cases initially identified on IHC but not on H&E were re-reviewed for viral inclusions on H&E: 3 had rare, but typical, inclusions; 3 had atypical inclusions; and 3 had no inclusions. CMV was not detected by H&E or IHC in 40 noncolitis controls. Of 10 steroid-refractory UC patients with CMV detected, 7 were refractory to cyclosporin or 6-mercaptopurine/azathioprine (70%) and 6 had undergone proctocolectomy (60%) prior to detection of the CMV. Two patients with recognized CMV infection were treated with gancyclovir, improved, and were able to taper off steroids and avoid proctocolectomy. This study provides evidence that unrecognized and therefore untreated CMV infection is significantly associated with steroid-refractory UC. Moreover, IHC is more sensitive than H&E for detection of CMV and should be considered as part of the routine evaluation of steroid-refractory UC patients, before proceeding with other medical or surgical therapy that may be unnecessary once the CMV is treated.
View details for Web of Science ID 000189316500009
View details for PubMedID 15104299
Detection of protein kinase-binding partners by the yeast two-hybrid analysis.
Methods in molecular biology (Clifton, N.J.)
2003; 233: 327-343
View details for PubMedID 12840519
RACK1: a novel substrate for the Src protein-tyrosine kinase
2002; 21 (50): 7619-7629
RACK1 is one of a group of PKC-interacting proteins collectively called RACKs (Receptors for Activated C-Kinases). Previously, we showed that RACK1 also interacts with the Src tyrosine kinase, and is an inhibitor of Src activity and cell growth. PKC activation induces the intracellular movement and co-localization of RACK1 and Src, and the tyrosine phosphorylation of RACK1. To determine whether RACK1 is a Src substrate, we assessed phosphorylation of RACK1 by various tyrosine kinases in vitro, and by kinase-active and inactive mutants of Src in vivo. We found that RACK1 is a Src substrate. Moreover, Src activity is necessary for both the tyrosine phosphorylation of RACK1 and the binding of RACK1 to Src's SH2 domain that occur following PKC activation. To identify the tyrosine(s) on RACK1 that is phosphorylated by Src, we generated and tested a series of RACK1 mutants. We found that Src phosphorylates RACK1 on Tyr 228 and/or Tyr 246, highly-conserved tyrosines located in the sixth WD repeat that interact with Src's SH2 domain. We think that RACK1 is an important Src substrate that signals downstream of growth factor receptor tyrosine kinases and is involved in the regulation of Src function and cell growth.
View details for DOI 10.1038/sj.onc.1206002
View details for Web of Science ID 000178756200002
View details for PubMedID 12400005
The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1
JOURNAL OF BIOLOGICAL CHEMISTRY
2001; 276 (23): 20346-20356
RACK1 is an intracellular receptor for the serine/ threonine protein kinase C. Previously, we demonstrated that RACK1 also interacts with the Src protein-tyrosine kinase. RACK1, via its association with these protein kinases, may play a key role in signal transduction. To further characterize the Src-RACK1 interaction and to analyze mechanisms by which cross-talk occurs between the two RACK1-linked signaling kinases, we identified sites on Src and RACK1 that mediate their binding, and factors that regulate their interaction. We found that the interaction of Src and RACK1 is mediated, in part, by the SH2 domain of Src and by phosphotyrosines in the sixth WD repeat of RACK1, and is enhanced by serum or platelet-derived growth factor stimulation, protein kinase C activation, and tyrosine phosphorylation of RACK1. To the best of our knowledge, this is the first report of tyrosine phosphorylation of a member of the WD repeat family of proteins. We think that tyrosine phosphorylation of these proteins is an important mechanism of signal transduction in cells.
View details for Web of Science ID 000169135100092
View details for PubMedID 11279199
The Shp-2 tyrosine phosphatase activates the Src tyrosine kinase by a non-enzymatic mechanism
1999; 18 (11): 1911-1920
Previously, we demonstrated that the Src tyrosine kinase interacts with the Shp-2 tyrosine phosphatase. To determine whether Shp-2 regulates Src kinase activity, we measured Src activity in cells overexpressing wild-type or catalytically-inactive C463S Shp-2. We observed a 2-3-fold increase in the specific activity of Src in both cell types and the increase did not appear to be due to dephosphorylation of Tyr 527 or phosphorylation of Tyr 416 on Src. Conversely, we observed a 2-3-fold decrease in the specific activity of Src when Shp-2 expression was inhibited. Using glutathione S-transferase-fusion proteins, we demonstrated that Shp-2 binds to the SH3 domain of Src. Our findings reveal that the Shp-2 tyrosine phosphatase can regulate the Src tyrosine kinase by a non-enzymatic mechanism. We also found that the phosphatase activity of Shp-2 immunoprecipitates is downregulated in cells transformed by Src or other proteins, and that Shp-2 preferentially associates with the membrane fraction of transformed cells. We suggest that membrane-association of Shp-2 is important for regulating Shp-2 activity.
View details for Web of Science ID 000079191300001
View details for PubMedID 10208413
RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of Src tyrosine kinases and growth of NIH 3T3 cells
MOLECULAR AND CELLULAR BIOLOGY
1998; 18 (6): 3245-3256
To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 microM) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G0/G1 than do wild-type cells. Prolongation of G0/G1 could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.
View details for Web of Science ID 000073628800015
View details for PubMedID 9584165
View details for PubMedCentralID PMC108906
- Intestinal cell growth control: Role of src tyrosine kinases GASTROENTEROLOGY 1998; 114 (6): 1335-1338
Src activation in malignant and premalignant epithelia of Barrett's esophagus
1997; 112 (2): 348-356
The neoplastic progression of Barrett's esophagus (BE) may involve genomic instability, inactivation of tumor suppressor genes, or activation of oncogenes. Because activation of Src tyrosine kinase occurs in malignant and premalignant epithelia of the colon, the aim of this study was to determine whether BE is associated with changes in Src expression and activity.Src expression and in vitro protein-tyrosine kinase activity in endoscopic tissue samples of BE and esophageal adenocarcinoma were measured and compared with expression and activity in normal esophagus and duodenum from the same patient. Src phosphorylation was assessed by immunoblotting using antiphosphotyrosine antibodies and two-dimensional tryptic phosphopeptide mapping.Src-specific activity was 3-4 fold higher in BE and 6-fold higher in esophageal adenocarcinoma than in control tissues. Different regions of BE from the same patient showed heterogeneity in Src activity compared with the uniform Src activity observed in different regions of normal esophagus and duodenum. In all tissues, Src kinase activity and protein were associated preferentially with the Triton X-100-soluble rather than-insoluble fraction. Immunoblotting and two-dimensional tryptic phosphopeptide mapping showed dephosphorylation of Src at Tyr527 in BE.Src is activated in BE, in part, because of dephosphorylation of Tyr527. Src activation and its heterogeneous expression occur before development of dysplasia or carcinoma in BE.
View details for Web of Science ID A1997WF18500008
View details for PubMedID 9024288
REGULATION OF THE SRC TYROSINE KINASE AND SYP TYROSINE PHOSPHATASE BY THEIR CELLULAR-ASSOCIATION
1995; 11 (10): 1955-1962
The specific activity of the Src tyrosine kinase is elevated in human colon carcinoma cells. To identify Src-binding proteins that might upregulate Src activity in these cells, a human colon carcinoma lambda gt11 expression library was screened with purified, 32P-labeled Src. The SH-PTP2 (Syp) tyrosine phosphatase was isolated and shown to associate with Src. In vitro studies demonstrated that: (1) transforming F527 Src phosphorylates Syp, and (2) Syp dephosphorylates Src at Tyr 527. Both events are known to upregulate enzyme activity. Others have shown that overexpression of the receptor tyrosine phosphatase alpha in rat embryo fibroblasts results in Src activation by dephosphorylation of Tyr 527, cell transformation and tumorigenesis. Thus, transmembrane tyrosine phosphatases may be involved in cell transformation exerting at least some of their effects through activation of Src. To the best of our knowledge, this is the first identification of an intracellular tyrosine, phosphatase which may activate Src by a similar mechanism.
View details for Web of Science ID A1995TF29700004
View details for PubMedID 7478513
SRC ACTIVITY INCREASES AND YES ACTIVITY DECREASES DURING MITOSIS OF HUMAN COLON-CARCINOMA CELLS
MOLECULAR AND CELLULAR BIOLOGY
1995; 15 (5): 2374-2382
Src and Yes protein-tyrosine kinase activities are elevated in malignant and premalignant tumors of the colon. To determine whether Src activity is elevated throughout the human colon carcinoma cell cycle as it is in polyomavirus middle T antigen- or F527 Src-transformed cells, and whether Yes activity, which is lower than that of Src in the carcinoma cells, is regulated differently, we measured their activities in cycling cells. We observed that the activities of both kinases were higher throughout all phases of the HT-29 colon carcinoma cell cycle than in corresponding phases of the fibroblast cycle. In addition, during mitosis of HT-29 cells, Src specific activity increased two- to threefold more, while Yes activity and abundance decreased threefold. The decreased steady-state protein levels of Yes during mitosis appeared to be due to both decreased synthesis and increased degradation of the protein. Inhibition of tyrosine but not serine/threonine phosphatases abolished the mitotic activation of Src. Mitotic Src was phosphorylated at novel serine and threonine sites and dephosphorylated at Tyr-527. Two cellular proteins (p160 and p180) were phosphorylated on tyrosine only during mitosis. Tyrosine phosphorylation of several other proteins decreased during mitosis. Thus, Src in HT-29 colon carcinoma cells, similar to Src complexed to polyomavirus middle T antigen or activated by mutation at Tyr-527, is highly active in all phases of the cell cycle. Moreover, Src activity further increases during mitosis, whereas Yes activity and abundance decrease. Thus, Src and Yes appear to be regulated differently during mitosis of HT-29 colon carcinoma cells.
View details for Web of Science ID A1995QU66500006
View details for PubMedID 7739521
View details for PubMedCentralID PMC230466
ELEVATED C-YES TYROSINE KINASE-ACTIVITY IN PREMALIGNANT LESIONS OF THE COLON
1995; 108 (1): 117-124
The cellular oncogene c-yes and its viral homologue v-yes (the transforming gene of Yamaguchi 73 and Esh avian sarcoma viruses) encode 62-kilodalton, cytoplasmic, membrane-associated, protein-tyrosine kinases. For the related Src kinase, a close correlation exists between elevated kinase activity and cell transformation. Previously, we observed elevated Yes activity in many human colon carcinomas. Colonic neoplasia provides an opportunity to study tumor progression because most carcinomas arise from adenomas, which in turn arise from normal epithelia. The malignant potential of adenomas varies with size, histology, and degree of dysplasia. Large adenomas (> or = 2 cm) with villous architecture and severe dysplasia are most likely to develop carcinoma.To determine whether Yes is activated in premalignant lesions of the colon, we measured its in vitro protein-tyrosine kinase activity in 21 colonic adenomas from 17 patients.Activity of Yes in adenomas at greatest risk for cancer was significantly greater (12- or 14-fold as measured by enolase or autophosphorylation, respectively) than activity in adjacent normal mucosa. Moreover, villous structure, large size (> or = 2 cm), or severe dysplasia correlated with elevated Yes activity.The activity of Yes is elevated in adenomas that are at greatest risk for developing cancer.
View details for Web of Science ID A1995PZ13500014
View details for PubMedID 7806032
ELEVATED C-SRC TYROSINE KINASE-ACTIVITY IN PREMALIGNANT EPITHELIA OF ULCERATIVE-COLITIS
JOURNAL OF CLINICAL INVESTIGATION
1994; 93 (2): 509-515
Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with a high incidence of colon cancer. Dysplasia is a precursor to carcinoma and a predictor of malignant potential; epithelia containing high-grade or severe dysplasia is most likely to develop cancer. The cellular oncogene c-src and its viral homologue v-src (the transforming gene of Rous sarcoma virus) encode 60-kD cytoplasmic, membrane-associated protein tyrosine kinases. For the viral protein or transforming mutants of the cellular protein (Src), a close correlation exists between elevated tyrosine kinase activity and malignant transformation of cells. Previously, we and others observed elevated Src activity in sporadic colon carcinomas and benign adenomas at greatest risk for developing cancer (those with large size, villous architecture, and/or severe dysplasia). Here we report that Src activity and protein abundance are also elevated in neoplastic UC epithelia. Activity is highest in malignant and severely dysplastic epithelia, and 6-10-fold higher in mildly dysplastic than in nondysplastic epithelia. Thus, Src activity is elevated in premalignant UC epithelia, which is at greatest risk for developing cancer. The data suggest that activation of the src proto-oncogene is an early event in the genesis of UC colon cancer.
View details for Web of Science ID A1994MY29600011
View details for PubMedID 7509341
View details for PubMedCentralID PMC293871
C-YES TYROSINE KINASE-ACTIVITY IN HUMAN COLON-CARCINOMA
1993; 8 (10): 2627-2635
To examine the role of Src-related proteins in human colon carcinoma we measured the tyrosine kinase activity of pp60c-src (Src), p62c-yes (Yes), p56lck (Lck), p59fyn (Fyn), p59hck (Hck), p56lyn (Lyn) and p55c-fgr (Fgr) from colonic cells. Yes activity, similar to that of Src, was 10-20 fold higher in three of five colon carcinoma cell lines and fivefold higher in 10 of 21 primary colon cancers than that in normal colonic cells. Lck activity was present in COLO 205 cells, otherwise Lck, Fyn, Hck, Lyn and Fgr activities were not detected in any of the carcinoma cell lines or cancers tested. Increased Yes activity, like that of Src, was due mostly to increased protein levels and not to an apparent decrease in phosphorylation of Tyr 537, the major mechanisms known to deregulate enzymatic activity. Only those colon carcinoma cell lines with elevated Src and/or Yes tyrosine kinase activity as measured in vitro had elevated levels of three tyrosine-phosphorylated proteins as measured in vivo. Thus, colon carcinoma cells contain active tyrosine kinases and/or inactive tyrosine phosphatases not present in normal colonic cells, and Src and Yes appear to be active kinases in the carcinoma cells. These data, together with those demonstrating decreased Src activity in fully differentiated enterocytes, suggest that down regulation of Src-related tyrosine kinases is important for differentiation, and/or deregulation of the kinases is important for growth and transformation of intestinal epithelial cells.
View details for Web of Science ID A1993LX34300003
View details for PubMedID 7690925
INTESTINAL CRYPT CELLS CONTAIN HIGHER LEVELS OF CYTOSKELETAL-ASSOCIATED PP60C-SRC PROTEIN TYROSINE KINASE-ACTIVITY THAN DO DIFFERENTIATED ENTEROCYTES
1993; 8 (4): 1033-1039
Undifferentiated crypt cells from chicken small intestine contain 15-fold higher levels of tyrosine-phosphorylated proteins than do differentiated enterocytes located at the villus apex. The tyrosine kinase activity and the tyrosine-phosphorylated proteins are associated with the Triton-insoluble cytoskeleton. To determine whether: (1) pp60c-src is an active tyrosine kinase in crypt cell cytoskeletons and (2) cytoskeletal-associated pp60c-src activity decreases as crypt cells differentiate, we isolated pp60c-src from subcellular fractions of cells along the crypt-villus axis of chicken small intestine and measured its protein kinase activity. We observed that pp60c-src activity in crypt cytoskeleton was higher (on average, fourfold as measured by enolase phosphorylation or sevenfold as measured by autophosphorylation) than that in cytoskeletons from differentiated enterocytes. Moreover, nearly 70% of pp60c-src activity in crypt cells, like that of pp60v-src, pp60c-src mutants with elevated kinase, activity or pp60v-src from activated platelets, localized to the cellular cytoskeleton. In contrast, less than 20% of pp60c-src activity in differentiated enterocytes, like that of kinase-inactive pp60v-src or pp60c-src from fibroblasts or resting platelets, associated with the cytoskeleton. Furthermore, in crypt cells, unlike differentiated enterocytes, cytoskeletal-associated pp60c-src appeared to have higher specific protein tyrosine kinase activity than did soluble pp60c-src. The data suggest that a kinase-active form of pp60c-src located in the cytoskeleton of crypt cells may be responsible for phosphorylating proteins on tyrosine and regulating growth and differentiation of the cells.
View details for Web of Science ID A1993KT22000027
View details for PubMedID 7681158
GENE STRUCTURE AND EXPRESSION IN COLORECTAL-CANCER
UNDERLYING MOLECULAR, CELLULAR, AND IMMUNOLOGICAL FACTORS IN CANCER AND AGING
1993; 330: 67-75
Colorectal cancer provides a unique model for the study of molecular changes that are associated with tumorigenesis. The cancer evolves as an apparent ordered sequence from a benign to a malignant lesion in histopathological recognizable stages. Since it is relatively easy to isolate tissue representing each of these stages, studies of molecular events associated with tumor progression are feasible. Such studies have shown that multiple changes in gene structure, expression and activity occur during tumorigenesis.
View details for Web of Science ID A1993BY65X00006
View details for PubMedID 7690179
THE HEPATITIS-B VIRUS-ENCODED TRANSCRIPTIONAL TRANSACTIVATOR HBX APPEARS TO BE A NOVEL PROTEIN SERINE THREONINE KINASE
1990; 63 (4): 687-695
To study the functional mechanism of the hepatitis B virus (HBV) X (hbx) gene product, we have expressed the hbx protein in E. coli and purified it by HPLC. The purified hbx protein was shown to be active in transactivating transcription directed by the LTR sequence of HIV-1. The hbx protein was found to have an intrinsic serine/threonine protein kinase activity. The hbx protein was detected in hepatitis B virions, and tryptic phosphopeptide maps of the hbx protein phosphorylated in the virion and of the in vitro phosphorylated bacterially expressed hbx protein were similar. Inactivation of the hbx protein by heat, protein-denaturing agents, or an ATP affinity analog, p-fluorosulfonylbenzoyl 5'-adenosine, resulted in loss of both protein kinase activity and trans-activation activity. These results suggest that the HBV-encoded trans-activator hbx is a novel protein kinase.
View details for Web of Science ID A1990EJ59000005
View details for PubMedID 2225072
A DNA REPLICATION-POSITIVE MUTANT OF SIMIAN VIRUS-40 THAT IS DEFECTIVE FOR TRANSFORMATION AND THE PRODUCTION OF INFECTIOUS VIRIONS
JOURNAL OF VIROLOGY
1990; 64 (6): 2912-2921
Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters.
View details for Web of Science ID A1990DD73500056
View details for PubMedID 2159552
View details for PubMedCentralID PMC249475
ACTIVATION OF THE PP60C-SRC PROTEIN-KINASE IS AN EARLY EVENT IN COLONIC CARCINOGENESIS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1990; 87 (2): 558-562
Colonic neoplasia provides an opportunity to study tumor progression because most carcinomas arise from adenomas (polyps), which, in turn, arise from normal epithelia. The malignant potential of adenomas varies with size, histology, and degree of dysplasia. Polyps that are less than 2 cm with villous architecture and severe dysplasia are most likely to contain carcinoma. Previous studies demonstrated that the in vitro protein-tyrosine kinase activity of pp60c-src from colon carcinomas is significantly higher than that from adjacent normal mucosa. Here we report that the protein kinase activity of pp60c-src is also elevated in colonic polyps. Activity is highest in malignant polyps and in greater than 2-cm benign polyps that contain villous structure and severe dysplasia. Thus, pp60c-src activation occurs in benign polyps that are at greatest risk for developing cancer. These data suggest that activation of the protooncogene product pp60c-src may be an important event in the genesis of human colon carcinoma.
View details for Web of Science ID A1990CK37800012
View details for PubMedID 2105487
EARLY ACTIVATION OF ENDOGENOUS PP60SRC KINASE-ACTIVITY DURING NEURONAL DIFFERENTIATION OF CULTURED HUMAN NEUROBLASTOMA-CELLS
MOLECULAR AND CELLULAR BIOLOGY
1990; 10 (1): 361-370
The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.
View details for Web of Science ID A1990CE81900040
View details for PubMedID 2136766
View details for PubMedCentralID PMC360755
PROPERTIES OF A SIMIAN-VIRUS 40 MUTANT-T ANTIGEN SUBSTITUTED IN THE HYDROPHOBIC REGION - DEFECTIVE ATPASE AND OLIGOMERIZATION ACTIVITIES AND ALTERED PHOSPHORYLATION ACCOMPANY AN INABILITY TO COMPLEX WITH CELLULAR P53
JOURNAL OF VIROLOGY
1989; 63 (8): 3362-3367
We have analyzed the biochemical properties of a nonviable simian virus 40 (SV40) mutant encoding a large T antigen (T) bearing an amino acid substitution (Pro-584-Leu) in its hydrophobic region. Mutant 5080 has an altered cell type specificity for transformation (transforming mouse C3H10T1/2 but not rat REF52 cells), is defective for viral DNA replication, and encodes a T that is unable to form a complex with the cellular p53 protein (K. Peden, A. Srinivasan, J. Farber, and J. Pipas, Virology 168:13-21, 1989). In this article, we show that 5080-transformed C3H10T1/2 cell lines express an altered T that is synthesized at a significantly higher rate but with a shorter half-life than normal T from wild-type SV40-transformed cells. 5080 T did not oligomerize beyond 5 to 10S in size compared with normal T, which oligomerized predominantly to 14 to 20S species. In addition, the 5080 T complex had significantly decreased ATPase activity and had a 10-fold-lower level of in vivo phosphorylation compared with that of normal T. Two-dimensional phosphopeptide analysis indicated several changes in the specific 32P labeling pattern, with altered phosphorylation occurring at both termini of the mutant protein compared with the wild-type T. Loss of p53 binding is therefore concomitant with changes in ATPase activity, oligomerization, stability, and in vivo phosphorylation of T and can be correlated with defective replication and restricted transformation functions. That so many biochemical changes are associated with a single substitution in the hydrophobic region of T is consistent with its importance in regulating higher-order structural and functional relationships in SV40 T.
View details for Web of Science ID A1989AF40600021
View details for PubMedID 2545911
View details for PubMedCentralID PMC250910
PP60C-SRC ACTIVATION IN HUMAN-COLON CARCINOMA
JOURNAL OF CLINICAL INVESTIGATION
1989; 83 (6): 2025-2033
We measured the in vitro protein-tyrosine kinase activity of pp60c-src from human colon carcinoma cell lines and tumors. The activity of pp60c-src from six of nine carcinoma cell lines was higher (on average, fivefold as measured by enolase phosphorylation, or eightfold as measured by autophosphorylation) than that of pp60c-src from normal colonic mucosal cells, or human or rodent fibroblasts. Similarly, the activity of pp60c-src from 13 of 21 primary colon carcinomas was five- or sevenfold higher than that of pp60c-src from normal colonic mucosa adjacent to the tumor. The increased pp60c-src activity did not result solely from an increase in the level of pp60c-src protein, suggesting the specific activity of the pp60c-src kinase is elevated in the tumor cells. pp60c-src from colon carcinoma cells and normal colonic mucosal cells was phosphorylated at similar sites. We used immunoblotting with antibodies to phosphotyrosine to identify substrates of protein-tyrosine kinases in colonic cells. Three phosphotyrosine-containing proteins were detected at significantly higher levels in most colon carcinoma cell lines than in normal colonic mucosal cells or human or rat fibroblasts. All colon carcinoma cell lines with elevated pp60c-src in vitro kinase activity, showed increased phosphorylation of proteins on tyrosine in vivo, suggesting the presence of an activated protein-tyrosine kinase(s).
View details for Web of Science ID A1989U951900031
View details for PubMedID 2498394
View details for PubMedCentralID PMC303927
PP60C-SRC EXPRESSION IN THE DEVELOPING RAT-BRAIN
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1988; 85 (10): 3348-3352
We have studied pp60c-src expression in the striatum, hippocampus, and cerebellum of the developing rat brain. In the striatum, pp60c-src protein kinase activity peaks during embryonic development and then declines in the adult. The peak activity occurs in the striatum on embryonic day 20 (E20) when it is 18- to 20-fold higher than the activity in fibroblasts and 4- to 5-fold higher than the activity in the striatum at E15 or in the adult striatum. In the hippocampal region, pp60c-src activity reaches a maximum shortly after birth but remains high throughout life. On postnatal day 2 (P2) the activity in the hippocampus is 9- to 13-fold higher than the activity in fibroblasts and twice as high as the activity in the hippocampus at E18. In the cerebellum, the kinase activity remains constant from E20 onward and is 6- to 10-fold higher than that observed in fibroblasts. The increase in pp60c-src kinase activity observed during the development of the striatum and hippocampus is due to an increase in the amount of pp60c-src protein and to an increase in the specific activity of the kinase. The increase in specific activity in these regions coincides with the peak periods of neurogenesis and neuronal growth. In the striatum, we have found that the increase in pp60c-src activity also parallels the increase observed in culture as embryonic striatal neurons differentiate. Taken together, our results are consonant with the idea that pp60c-src is the product of a developmentally regulated gene that is important for the differentiation and/or the continuing function of neurons.
View details for Web of Science ID A1988N475300018
View details for PubMedID 2453056
View details for PubMedCentralID PMC280206
ALTERATIONS IN PP60C-SRC ACCOMPANY DIFFERENTIATION OF NEURONS FROM RAT EMBRYO STRIATUM
MOLECULAR AND CELLULAR BIOLOGY
1987; 7 (5): 1830-1840
Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60c-src. The 60-kilodalton (kDa) form appeared similar to pp60c-src from cultured rat fibroblasts or astrocytes. The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60c-src was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60c-src was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60c-src population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60c-src and the increase in specific protein kinase activity may be important for neuronal differentiation.
View details for Web of Science ID A1987H060600030
View details for PubMedID 2439897
View details for PubMedCentralID PMC365286
CDNA CLONING WITH A RETROVIRUS EXPRESSION VECTOR - GENERATION OF A PP60C-SRC CDNA CLONE
JOURNAL OF VIROLOGY
1987; 61 (5): 1731-1734
We used a murine retroviral expression vector, containing a genomic clone of the chicken c-src gene, a bacterial origin of replication, and a selectable marker, to remove 10 introns from the c-src gene. All 10 introns were removed accurately, and no mutations were introduced. The processed gene encoded a functional pp60c-src protein tyrosine kinase.
View details for Web of Science ID A1987G923200064
View details for PubMedID 2437323
View details for PubMedCentralID PMC254166
CELL-TRANSFORMATION BY PP60C-SRC MUTATED IN THE CARBOXY-TERMINAL REGULATORY DOMAIN
1987; 49 (1): 83-91
We introduced two mutations into the carboxy-terminal regulatory region of chicken pp60c-src. One, F527, replaces tyrosine 527 with phenylalanine. The other, Am517, produces a truncated pp60c-src protein lacking the 17 carboxy-terminal amino acids. Both mutant proteins were phosphorylated at tyrosine 416 in vivo. The specific activity of the Am517 mutant protein kinase was similar to that of wild-type pp60c-src whereas that of the F527 mutant was 5- to 10-fold higher. Both mutant c-src genes induced focus formation on NIH 3T3 cells, but the foci appeared at lower frequency, and were smaller than foci induced by polyoma middle tumor antigen (mT). The wild-type or F527 pp60c-src formed a complex with mT, whereas the Am517 pp60c-src did not. The results suggest that one, inability to phosphorylate tyrosine 527 increases pp60c-src protein kinase activity and transforming ability; two, transformation by mT involves other events besides lack of phosphorylation at tyrosine 527 of pp60c-src; three, activation of the pp60c-src protein kinase may not be required for transformation by the Am517 mutant; and four, the carboxyl terminus of pp60c-src appears to be required for association with mT.
View details for Web of Science ID A1987G882600011
View details for PubMedID 3103927
INHIBITION OF ATP LEVELS BY QUINIDINE IN A HUMAN COLONIC EPITHELIAL-CELL LINE
AMERICAN JOURNAL OF PHYSIOLOGY
1986; 250 (6): G806-G813
The influence of quinidine, a putative K+ channel blocker, on Cl- secretion induced by vasoactive intestinal polypeptide (VIP) was investigated. Quinidine inhibited Cl- secretion induced by VIP in T84 cell monolayers. A similar inhibitory effect of quinidine on Cl- secretion was observed in an isolated human colon. However, in the isolated human colon, which absorbs Na+ avidly, inhibition of Na+ absorption predominated. In the T84 cell, the half-maximal inhibition by quinidine occurred at 60 microM, while 300 microM almost completely inhibited the VIP-induced Cl- secretion. Mucosal addition of quinidine was at least equally effective compared with serosal addition, suggesting that quinidine acts on the apical membrane or intracellularly. Quinidine had little or no effect on VIP-stimulated Cl- efflux in the first 15 min after its addition, suggesting that blockage of the Cl- exit pathway on the apical membrane is an unlikely mechanism. Similarly, quinidine did not inhibit the K+-recycling mechanism on the basolateral membrane in the first 15 min after its addition. The initial inhibitory action of quinidine corresponded better with its ability to decrease cellular ATP levels. Our study suggests that the depletion of cellular ATP levels may explain the initial inhibitory action of quinidine on electrolyte transport in the intestine, while the late effect is multifactorial.
View details for Web of Science ID A1986D017600052
View details for PubMedID 3013024
ALTERED SITES OF TYROSINE PHOSPHORYLATION IN PP60C-SRC ASSOCIATED WITH POLYOMAVIRUS MIDDLE TUMOR-ANTIGEN
MOLECULAR AND CELLULAR BIOLOGY
1986; 6 (5): 1562-1570
We characterized the tyrosine phosphorylation sites of free pp60c-src and of pp60c-src associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60c-src were different, both in vitro and in vivo. Free pp60c-src was phosphorylated in vitro at a single site, tyrosine 416. pp60c-src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60c-src in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60c-src associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60c-src associated with mT in transformed cells are identical to those of pp60v-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60c-src associated with mT may play a role in the enhancement of the pp60c-src protein kinase activity and in cell transformation by polyomavirus.
View details for Web of Science ID A1986C111200024
View details for PubMedID 2431281
View details for PubMedCentralID PMC367682
TYR527 IS PHOSPHORYLATED IN PP60C-SRC - IMPLICATIONS FOR REGULATION
1986; 231 (4744): 1431-1434
The Rous sarcoma virus oncogene product, pp60v-src, transforms cultured fibroblasts but its corresponding proto-oncogene product, pp60c-src, does not. Both proteins are known to be protein-tyrosine kinases. Published results suggest that the kinase activity of pp60c-src is inhibited relative to that of pp60v-src, due perhaps to phosphorylation of a tyrosine in pp60c-src that is not phosphorylated in pp60v-src. In this study, it was observed that the tyrosine phosphorylated in pp60c-src is Tyr527, six residues from the COOH-terminus of the protein. The region of pp60c-src from residue 515 to the COOH-terminus, including Tyr527, has been replaced with a different sequence in pp60v-src. Thus, the increase in transforming ability and kinase activity that occurred in the genesis of pp60v-src may have resulted from the loss of a tyrosine involved in negative regulation.
View details for Web of Science ID A1986A391100037
View details for PubMedID 2420005
STRUCTURAL AND FUNCTIONAL MODIFICATION OF PP60C-SRC ASSOCIATED WITH POLYOMA MIDDLE TUMOR-ANTIGEN FROM INFECTED OR TRANSFORMED-CELLS
MOLECULAR AND CELLULAR BIOLOGY
1985; 5 (10): 2647-2652
The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.
View details for Web of Science ID A1985ARN8800020
View details for PubMedID 2426573
View details for PubMedCentralID PMC367001
SYNERGISTIC ACTION OF CYCLIC ADENOSINE MONOPHOSPHATE-MEDIATED AND CALCIUM-MEDIATED CHLORIDE SECRETION IN A COLONIC EPITHELIAL-CELL LINE
JOURNAL OF CLINICAL INVESTIGATION
1985; 76 (5): 1837-1842
Vasoactive intestinal polypeptide (VIP) and the calcium ionophore A23187 caused dose-dependent changes in the potential difference and the short circuit current (Isc) across confluent T84 cell monolayers mounted in modified Ussing chambers. Both VIP and A23187 stimulated net chloride secretion without altering sodium transport. Net chloride secretion accounted for the increase in Isc. When A23187 was tested in combination with VIP, net chloride secretion was significantly greater than predicted from the calculated sum of their individual responses indicating a synergistic effect. VIP increased cellular cyclic AMP (cAMP) production in a dose-dependent manner, whereas A23187 had no effect on cellular cAMP. We then determined whether VIP and A23187 activated different transport pathways. Earlier studies suggest that VIP activates a basolaterally localized, barium-sensitive potassium channel as well as an apically localized chloride conductance pathway. In this study, stimulation of basolateral membrane potassium efflux by A23187 was documented by preloading the monolayers with 86Rb+. Stimulation of potassium efflux by A23187 was additive to the VIP-stimulated potassium efflux. By itself, 0.3 microM A23187 did not alter transepithelial chloride permeability, and its stimulation of basolateral membrane potassium efflux caused only a relatively small amount of chloride secretion. However, in the presence of an increased transepithelial chloride permeability induced by VIP, the effectiveness of A23187 on chloride secretion was greatly augmented. Our studies suggest that cAMP and calcium each activate basolateral potassium channels, but cAMP also activates an apically localized chloride channel. Synergism results from cooperative interaction of potassium channels and the chloride channel.
View details for Web of Science ID A1985AUM6100017
View details for PubMedID 2997291
View details for PubMedCentralID PMC424220
Electrolyte transport in a human colonic cell line: regulation by cAMP and calcium.
Kroc Foundation series
1984; 17: 65-75
View details for PubMedID 6096515
PELIOSIS HEPATIS - DIAGNOSIS BY MAGNIFICATION WEDGED HEPATIC VENOGRAPHY
1984; 150 (3): 647-649
Benign hepatic tumors and peliosis hepatis developed in a patient who had received androgen therapy for three years. The tumors were identified by arteriography. Peliosis hepatis was diagnosed by wedged hepatic venography: to our knowledge, this method has not been previously used to diagnose this condition. Wedged venography, performed four months after withdrawal of androgen therapy and after liver function had nearly returned to normal, demonstrated morphologic improvement as well. On the basis of this case and other histologic descriptions of peliosis hepatis, we believe that wedged hepatic venography is a simple, sensitive, and specific diagnostic modality.
View details for Web of Science ID A1984SD43800004
View details for PubMedID 6695062
MANAGEMENT OF ACUTE DIARRHEA
JOURNAL OF CLINICAL GASTROENTEROLOGY
1983; 5 (6): 523-534
Acute diarrhea in adults is usually caused by bacterial infection, parasitic infestation, food poisoning, or drugs. In this review, we summarize the pros and cons of medical therapies, point out certain areas of controversy, and propose a rationale for early intervention. Discussion is limited to the use of antidiarrheal agents and antibiotics in acute infectious diarrhea.
View details for Web of Science ID A1983RW05500008
View details for PubMedID 6363516
- INNOMINATE VEIN OBSTRUCTION CAUSED BY INTRA-THORACIC GOITER WESTERN JOURNAL OF MEDICINE 1981; 135 (3): 235-238
- EFFECT OF MORPHINE ON SEROTONIN RELEASE FROM MYENTERIC PLEXUS OF GUINEA-PIG JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 1974; 190 (3): 420-430
- REVERSIBILITY OF MORPHINE-TOLERANCE AND DEPENDENCE IN GUINEA-PIG BRAIN AND MYENTERIC PLEXUS NATURE 1974; 251 (5473): 329-331