All Publications

  • Gene-teratogen interactions influence the penetrance of birth defects by altering Hedgehog signaling strength. Development (Cambridge, England) Kong, J. H., Young, C. B., Pusapati, G. V., Espinoza, F. H., Patel, C. B., Beckert, F., Ho, S., Patel, B. B., Gabriel, G. C., Aravind, L., Bazan, J. F., Gunn, T. M., Lo, C. W., Rohatgi, R. 2021


    Birth defects result from interactions between genetic and environmental factors, but the mechanisms remain poorly understood. We find that mutations and teratogens interact in predictable ways to cause birth defects by changing target cell sensitivity to Hedgehog (Hh) ligands. These interactions converge on a membrane protein complex, the MMM complex, that promotes degradation of the Hh transducer Smoothened (SMO). Deficiency of the MMM component MOSMO results in elevated SMO and increased Hh signaling, causing multiple birth defects. In utero exposure to a teratogen that directly inhibits SMO reduces the penetrance and expressivity of birth defects in Mosmo-/- embryos. Additionally, tissues that develop normally in Mosmo-/- embryos are refractory to the teratogen. Thus, changes in the abundance of the protein target of a teratogen can change birth defect outcomes by quantitative shifts in Hh signaling. Consequently, small molecules that re-calibrate signaling strength could be harnessed to rescue structural birth defects.

    View details for DOI 10.1242/dev.199867

    View details for PubMedID 34486668

  • Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling EMBO MOLECULAR MEDICINE Bosakova, M., Abraham, S. P., Nita, A., Hruba, E., Buchtova, M., Taylor, S., Duran, I., Martin, J., Svozilova, K., Barta, T., Varecha, M., Balek, L., Kohoutek, J., Radaszkiewicz, T., Pusapati, G., Bryja, V., Rush, E. T., Thiffault, I., Nickerson, D. A., Bamshad, M. J., Rohatgi, R., Cohn, D. H., Krakow, D., Krejci, P. 2020
  • A Membrane-Tethered Ubiquitination Pathway Regulates Hedgehog Signaling and Heart Development. Developmental cell Kong, J. H., Young, C. B., Pusapati, G. V., Patel, C. B., Ho, S. n., Krishnan, A. n., Lin, J. I., Devine, W. n., Moreau de Bellaing, A. n., Athni, T. S., Aravind, L. n., Gunn, T. M., Lo, C. W., Rohatgi, R. n. 2020


    The etiology of congenital heart defects (CHDs), which are among the most common human birth defects, is poorly understood because of its complex genetic architecture. Here, we show that two genes implicated in CHDs, Megf8 and Mgrn1, interact genetically and biochemically to regulate the strength of Hedgehog signaling in target cells. MEGF8, a transmembrane protein, and MGRN1, a RING superfamily E3 ligase, assemble to form a receptor-like ubiquitin ligase complex that catalyzes the ubiquitination and degradation of the Hedgehog pathway transducer Smoothened. Homozygous Megf8 and Mgrn1 mutations increased Smoothened abundance and elevated sensitivity to Hedgehog ligands. While mice heterozygous for loss-of-function Megf8 or Mgrn1 mutations were normal, double heterozygous embryos exhibited an incompletely penetrant syndrome of CHDs with heterotaxy. Thus, genetic interactions can arise from biochemical mechanisms that calibrate morphogen signaling strength, a conclusion broadly relevant for the many human diseases in which oligogenic inheritance is emerging as a mechanism for heritability.

    View details for DOI 10.1016/j.devcel.2020.08.012

    View details for PubMedID 32966817

  • R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling. eLife Dubey, R. n., van Kerkhof, P. n., Jordens, I. n., Malinauskas, T. n., Pusapati, G. V., McKenna, J. K., Li, D. n., Carette, J. E., Ho, M. n., Siebold, C. n., Maurice, M. n., Lebensohn, A. M., Rohatgi, R. n. 2020; 9


    R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT/β-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSPO3 using a combination of ligand mutagenesis and ligand engineering. Mutations in RSPO3 residues predicted to contact HSPGs impair its signaling capacity. Conversely, the HSPG-binding domains of RSPO3 can be entirely replaced with an antibody that recognizes heparan sulfate (HS) chains attached to multiple HSPGs without diminishing WNT-potentiating activity in cultured cells and intestinal organoids. A genome-wide screen for mediators of RSPO3 signaling in cells lacking LGRs 4, 5 and 6 failed to reveal other receptors. We conclude that HSPGs are RSPO co-receptors that potentiate WNT signaling in the presence and absence of LGRs.

    View details for DOI 10.7554/eLife.54469

    View details for PubMedID 32432544

  • Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling. EMBO molecular medicine Bosakova, M. n., Abraham, S. P., Nita, A. n., Hruba, E. n., Buchtova, M. n., Taylor, S. P., Duran, I. n., Martin, J. n., Svozilova, K. n., Barta, T. n., Varecha, M. n., Balek, L. n., Kohoutek, J. n., Radaszkiewicz, T. n., Pusapati, G. V., Bryja, V. n., Rush, E. T., Thiffault, I. n., Nickerson, D. A., Bamshad, M. J., Rohatgi, R. n., Cohn, D. H., Krakow, D. n., Krejci, P. n. 2020; 12 (11): e11739


    Mutations in genes affecting primary cilia cause ciliopathies, a diverse group of disorders often affecting skeletal development. This includes Jeune syndrome or asphyxiating thoracic dystrophy (ATD), an autosomal recessive skeletal disorder. Unraveling the responsible molecular pathology helps illuminate mechanisms responsible for functional primary cilia. We identified two families with ATD caused by loss-of-function mutations in the gene encoding adrenergic receptor kinase 1 (ADRBK1 or GRK2). GRK2 cells from an affected individual homozygous for the p.R158* mutation resulted in loss of GRK2, and disrupted chondrocyte growth and differentiation in the cartilage growth plate. GRK2 null cells displayed normal cilia morphology, yet loss of GRK2 compromised cilia-based signaling of Hedgehog (Hh) pathway. Canonical Wnt signaling was also impaired, manifested as a failure to respond to Wnt ligand due to impaired phosphorylation of the Wnt co-receptor LRP6. We have identified GRK2 as an essential regulator of skeletogenesis and demonstrate how both Hh and Wnt signaling mechanistically contribute to skeletal ciliopathies.

    View details for DOI 10.15252/emmm.201911739

    View details for PubMedID 33200460

    View details for PubMedCentralID PMC7645380

  • Discovery of gene regulatory elements through a new bioinformatics analysis of haploid genetic screens. PloS one Patel, B. B., Lebensohn, A. M., Pusapati, G. V., Carette, J. E., Salzman, J. n., Rohatgi, R. n. 2019; 14 (1): e0198463


    The systematic identification of regulatory elements that control gene expression remains a challenge. Genetic screens that use untargeted mutagenesis have the potential to identify protein-coding genes, non-coding RNAs and regulatory elements, but their analysis has mainly focused on identifying the former two. To identify regulatory elements, we conducted a new bioinformatics analysis of insertional mutagenesis screens interrogating WNT signaling in haploid human cells. We searched for specific patterns of retroviral gene trap integrations (used as mutagens in haploid screens) in short genomic intervals overlapping with introns and regions upstream of genes. We uncovered atypical patterns of gene trap insertions that were not predicted to disrupt coding sequences, but caused changes in the expression of two key regulators of WNT signaling, suggesting the presence of cis-regulatory elements. Our methodology extends the scope of haploid genetic screens by enabling the identification of regulatory elements that control gene expression.

    View details for PubMedID 30695034

  • Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling. eLife Kinnebrew, M. n., Iverson, E. J., Patel, B. B., Pusapati, G. V., Kong, J. H., Johnson, K. A., Luchetti, G. n., Eckert, K. M., McDonald, J. G., Covey, D. F., Siebold, C. n., Radhakrishnan, A. n., Rohatgi, R. n. 2019; 8


    Previously we proposed that transmission of the Hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

    View details for DOI 10.7554/eLife.50051

    View details for PubMedID 31657721

  • CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog. Developmental cell Pusapati, G. V., Kong, J. H., Patel, B. B., Krishnan, A. n., Sagner, A. n., Kinnebrew, M. n., Briscoe, J. n., Aravind, L. n., Rohatgi, R. n. 2018; 44 (1): 113–29.e8


    To uncover regulatory mechanisms in Hedgehog (Hh) signaling, we conducted genome-wide screens to identify positive and negative pathway components and validated top hits using multiple signaling and differentiation assays in two different cell types. Most positive regulators identified in our screens, including Rab34, Pdcl, and Tubd1, were involved in ciliary functions, confirming the central role for primary cilia in Hh signaling. Negative regulators identified included Megf8, Mgrn1, and an unannotated gene encoding a tetraspan protein we named Atthog. The function of these negative regulators converged on Smoothened (SMO), an oncoprotein that transduces the Hh signal across the membrane. In the absence of Atthog, SMO was stabilized at the cell surface and concentrated in the ciliary membrane, boosting cell sensitivity to the ligand Sonic Hedgehog (SHH) and consequently altering SHH-guided neural cell-fate decisions. Thus, we uncovered genes that modify the interpretation of morphogen signals by regulating protein-trafficking events in target cells.

    View details for PubMedID 29290584

    View details for PubMedCentralID PMC5792066

  • Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling. Nature communications Hong, S. R., Wang, C. L., Huang, Y. S., Chang, Y. C., Chang, Y. C., Pusapati, G. V., Lin, C. Y., Hsu, N. n., Cheng, H. C., Chiang, Y. C., Huang, W. E., Shaner, N. C., Rohatgi, R. n., Inoue, T. n., Lin, Y. C. 2018; 9 (1): 1732


    Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here, we develop a method to rapidly deplete tubulin glutamylation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

    View details for PubMedID 29712905

    View details for PubMedCentralID PMC5928066

  • G protein-coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog. Science signaling Pusapati, G. V., Kong, J. H., Patel, B. B., Gouti, M. n., Sagner, A. n., Sircar, R. n., Luchetti, G. n., Ingham, P. W., Briscoe, J. n., Rohatgi, R. n. 2018; 11 (516)


    The morphogen Sonic Hedgehog (SHH) patterns tissues during development by directing cell fates in a concentration-dependent manner. The SHH signal is transmitted across the membrane of target cells by the heptahelical transmembrane protein Smoothened (SMO), which activates the GLI family of transcription factors through a mechanism that is undefined in vertebrates. Using CRISPR-edited null alleles and small-molecule inhibitors, we systematically analyzed the epistatic interactions between SMO and three proteins implicated in SMO signaling: the heterotrimeric G protein subunit GαS, the G protein-coupled receptor kinase 2 (GRK2), and the GαS-coupled receptor GPR161. Our experiments uncovered a signaling mechanism that modifies the sensitivity of target cells to SHH and consequently changes the shape of the SHH dose-response curve. In both fibroblasts and spinal neural progenitors, the loss of GPR161, previously implicated as an inhibitor of basal SHH signaling, increased the sensitivity of target cells across the entire spectrum of SHH concentrations. Even in cells lacking GPR161, GRK2 was required for SHH signaling, and Gαs, which promotes the activation of protein Kinase A (PKA), antagonized SHH signaling. We propose that the sensitivity of target cells to Hedgehog morphogens, and the consequent effects on gene expression and differentiation outcomes, can be controlled by signals from G protein-coupled receptors that converge on Gαsand PKA.

    View details for PubMedID 29438014

    View details for PubMedCentralID PMC5828112

  • Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision. Cell Phua, S. C., Chiba, S., Suzuki, M., Su, E., Roberson, E. C., Pusapati, G. V., Setou, M., Rohatgi, R., Reiter, J. F., Ikegami, K., Inoue, T. 2017; 168 (1-2): 264-279 e15


    The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

    View details for DOI 10.1016/j.cell.2016.12.032

    View details for PubMedID 28086093

  • An essential role for Grk2 in Hedgehog signalling downstream of Smoothened EMBO REPORTS Zhao, Z., Lee, R. T., Pusapati, G. V., Iyu, A., Rohatgi, R., Ingham, P. W. 2016; 17 (5): 739-752


    The G-protein-coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock-down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP-dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase-dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C-terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho-null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.

    View details for Web of Science ID 000375617400015

    View details for PubMedID 27113758

  • Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity. Nature Carmi, Y., Spitzer, M. H., Linde, I. L., Burt, B. M., Prestwood, T. R., Perlman, N., Davidson, M. G., Kenkel, J. A., Segal, E., Pusapati, G. V., Bhattacharya, N., Engleman, E. G. 2015; 521 (7550): 99-104


    Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.

    View details for DOI 10.1038/nature14424

    View details for PubMedID 25924063

    View details for PubMedCentralID PMC4877172

  • Location, location, and location: compartmentalization of Hedgehog signaling at primary cilia. EMBO journal Pusapati, G. V., Rohatgi, R. 2014; 33 (17): 1852-1854

    View details for DOI 10.15252/embj.201489294

    View details for PubMedID 25037564

  • EFCAB7 and IQCE Regulate Hedgehog Signaling by Tethering the EVC-EVC2 Complex to the Base of Primary Cilia DEVELOPMENTAL CELL Pusapati, G. V., Hughes, C. E., Dorn, K. V., Zhang, D., Sugianto, P., Aravind, L., Rohatgi, R. 2014; 28 (5): 483-496


    The Hedgehog (Hh) pathway depends on primary cilia in vertebrates, but the signaling machinery within cilia remains incompletely defined. We report the identification of a complex between two ciliary proteins, EFCAB7 and IQCE, which positively regulates the Hh pathway. The EFCAB7-IQCE module anchors the EVC-EVC2 complex in a signaling microdomain at the base of cilia. EVC and EVC2 genes are mutated in Ellis van Creveld and Weyers syndromes, characterized by impaired Hh signaling in skeletal, cardiac, and orofacial tissues. EFCAB7 binds to a C-terminal disordered region in EVC2 that is deleted in Weyers patients. EFCAB7 depletion mimics the Weyers cellular phenotype-the mislocalization of EVC-EVC2 within cilia and impaired activation of the transcription factor GLI2. Evolutionary analysis suggests that emergence of these complexes might have been important for adaptation of an ancient organelle, the cilium, for an animal-specific signaling network.

    View details for DOI 10.1016/j.devcel.2014.01.021

    View details for Web of Science ID 000333405600004

    View details for PubMedID 24582806

    View details for PubMedCentralID PMC4027042

  • A novel splice variant of calcium and integrin-binding protein 1 mediates protein kinase D2-stimulated tumour growth by regulating angiogenesis. Oncogene Armacki, M., Joodi, G., Nimmagadda, S. C., De Kimpe, L., Pusapati, G. V., Vandoninck, S., Van Lint, J., Illing, A., Seufferlein, T. 2014; 33 (9): 1167-1180


    Protein kinase D2 (PKD2) is a member of the PKD family of serine/threonine kinases, a subfamily of the CAMK super-family. PKDs have a critical role in cell motility, migration and invasion of cancer cells. Expression of PKD isoforms is deregulated in various tumours and PKDs, in particular PKD2, have been implicated in the regulation of tumour angiogenesis. In order to further elucidate the role of PKD2 in tumours, we investigated the signalling context of this kinase by performing an extensive substrate screen by in vitro expression cloning (IVEC). We identified a novel splice variant of calcium and integrin-binding protein 1, termed CIB1a, as a potential substrate of PKD2. CIB1 is a widely expressed protein that has been implicated in angiogenesis, cell migration and proliferation, all important hallmarks of cancer, and CIB1a was found to be highly expressed in various cancer cell lines. We identify Ser(118) as the major PKD2 phosphorylation site in CIB1a and show that PKD2 interacts with CIB1a via its alanine and proline-rich domain. Furthermore, we confirm that CIB1a is indeed a substrate of PKD2 also in intact cells using a phosphorylation-specific antibody against CIB1a-Ser(118). Functional analysis of PKD2-mediated CIB1a phosphorylation revealed that on phosphorylation, CIB1a mediates tumour cell invasion, tumour growth and angiogenesis by mediating PKD-induced vascular endothelial growth factor secretion by the tumour cells. Thus, CIB1a is a novel mediator of PKD2-driven carcinogenesis and a potentially interesting therapeutic target.Oncogene advance online publication, 18 March 2013; doi:10.1038/onc.2013.43.

    View details for DOI 10.1038/onc.2013.43

    View details for PubMedID 23503467

  • Ric1-Rgp1 Complex Is a Guanine Nucleotide Exchange Factor for the Late Golgi Rab6A GTPase and an Effector of the Medial Golgi Rab33B GTPase JOURNAL OF BIOLOGICAL CHEMISTRY Pusapati, G. V., Luchetti, G., Pfeffer, S. R. 2012; 287 (50): 42129-42137


    Rab GTPases are master regulators of membrane trafficking events and template the directionality of protein transport through the secretory and endocytic pathways. Certain Rabs recruit the guanine nucleotide exchange factor (GEF) that activates a subsequent acting Rab protein in a given pathway; this process has been termed a Rab cascade. We show here that the medial Golgi-localized Rab33B GTPase has the potential to link functionally to the late Golgi, Rab6 GTPase, by its capacity for association with Ric1 and Rgp1 proteins. In yeast, Ric1p and Rgp1p form a complex that catalyzes guanine nucleotide exchange by Ypt6p, the Rab6 homolog. Human Ric1 and Rgp1 both bind Rab6A with preference for the GDP-bound conformation, characteristic of a GEF. Nevertheless, both Ric1 and Rgp1 proteins are needed to catalyze nucleotide exchange on Rab6A protein. Ric1 and Rgp1 form a complex, but unlike their yeast counterparts, most of the subunits are not associated, and most of the proteins are cytosolic. Loss of Ric1 or Rgp1 leads to destabilization of Rab6, concomitant with a block in Rab6-dependent retrograde transport of mannose 6-phosphate receptors to the Golgi. The C terminus of Ric1 protein contains a distinct binding site for Rab33B-GTP, supporting the existence of a Rab cascade between the medial and trans Golgi. This study thus identifies a GEF for Rab6A in human cells.

    View details for DOI 10.1074/jbc.M112.414565

    View details for Web of Science ID 000312103000051

    View details for PubMedID 23091056

    View details for PubMedCentralID PMC3516758

  • RUTBC2 Protein, a Rab9A Effector and GTPase-activating Protein for Rab36 JOURNAL OF BIOLOGICAL CHEMISTRY Nottingham, R. M., Pusapati, G. V., Ganley, I. G., Barr, F. A., Lambright, D. G., Pfeffer, S. R. 2012; 287 (27): 22740-22748


    Rab GTPases regulate vesicle budding, motility, docking, and fusion. In cells, their cycling between active, GTP-bound states and inactive, GDP-bound states is regulated by the action of opposing enzymes called guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). The substrates for most RabGAPs are unknown, and the potential for cross-talk between different membrane trafficking pathways remains uncharted territory. Rab9A and its effectors regulate recycling of mannose 6-phosphate receptors from late endosomes to the trans Golgi network. We show here that RUTBC2 is a TBC domain-containing protein that binds to Rab9A specifically both in vitro and in cultured cells but is not a GAP for Rab9A. Biochemical screening of Rab protein substrates for RUTBC2 revealed highest GAP activity toward Rab34 and Rab36. In cells, membrane-associated RUTBC2 co-localizes with Rab36, and expression of wild type RUTBC2, but not the catalytically inactive, RUTBC2 R829A mutant, decreases the amount of membrane-associated Rab36 protein. These data show that RUTBC2 can act as a Rab36 GAP in cells and suggest that RUTBC2 links Rab9A function to Rab36 function in the endosomal system.

    View details for DOI 10.1074/jbc.M112.362558

    View details for Web of Science ID 000306495000031

    View details for PubMedID 22637480

    View details for PubMedCentralID PMC3391118

  • Protein Kinase D Regulates RhoA Activity via Rhotekin Phosphorylation JOURNAL OF BIOLOGICAL CHEMISTRY Pusapati, G. V., Eiseler, T., Rykx, A., Vandoninck, S., Derua, R., Waelkens, E., Van Lint, J., von Wichert, G., Seufferlein, T. 2012; 287 (12): 9473-9483


    The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin.

    View details for DOI 10.1074/jbc.M112.339564

    View details for Web of Science ID 000301797800076

    View details for PubMedID 22228765

  • Protein kinase D2 is a novel regulator of glioblastoma growth and tumor formation NEURO-ONCOLOGY Azoitei, N., Kleger, A., Schoo, N., Thal, D. R., Brunner, C., Pusapati, G. V., Filatova, A., Genze, F., Moeller, P., Acker, T., Kuefer, R., Van Lint, J., Baust, H., Adler, G., Seufferlein, T. 2011; 13 (7): 710-724


    Glioblastoma multiforme, a highly aggressive tumor of the central nervous system, has a dismal prognosis that is due in part to its resistance to radio- and chemotherapy. The protein kinase C (PKC) family of serine threonine kinases has been implicated in the formation and proliferation of glioblastoma multiforme. Members of the protein kinase D (PKD) family, which consists of PKD1, -2 and, -3, are prominent downstream targets of PKCs and could play a major role in glioblastoma growth. PKD2 was highly expressed in both low-grade and high-grade human gliomas. The number of PKD2-positive tumor cells increased with glioma grading (P < .001). PKD2 was also expressed in CD133-positive glioblastoma stem cells and various glioblastoma cell lines in which the kinase was found to be constitutively active. Inhibition of PKDs by pharmacological inhibitors resulted in substantial inhibition of glioblastoma proliferation. Furthermore, specific depletion of PKD2 by siRNA resulted in a marked inhibition of anchorage-dependent and -independent proliferation and an accumulation of glioblastoma cells in G0/G1, accompanied by a down-regulation of cyclin D1 expression. In addition, PKD2-depleted glioblastoma cells exhibited substantially reduced tumor formation in vivo on chicken chorioallantoic membranes. These findings identify PKD2 as a novel mediator of glioblastoma cell growth in vitro and in vivo and thereby as a potential therapeutic target for this devastating disease.

    View details for DOI 10.1093/neuonc/nor084

    View details for Web of Science ID 000293170900003

    View details for PubMedID 21727210

  • Protein Kinase D2 Is an Essential Regulator of Murine Myoblast Differentiation PLOS ONE Kleger, A., Loebnitz, C., Pusapati, G. V., Armacki, M., Mueller, M., Tuempel, S., Illing, A., Hartmann, D., Brunner, C., Liebau, S., Rudolph, K. L., Adler, G., Seufferlein, T. 2011; 6 (1)


    Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC) family. The protein kinase D (PKD) isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12) as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.

    View details for DOI 10.1371/journal.pone.0014599

    View details for Web of Science ID 000286663900004

    View details for PubMedID 21298052

  • Modulation of Calcium-Activated Potassium Channels Induces Cardiogenesis of Pluripotent Stem Cells and Enrichment of Pacemaker-Like Cells CIRCULATION Kleger, A., Seufferlein, T., Malan, D., Tischendorf, M., Storch, A., Wolheim, A., Latz, S., Protze, S., Porzner, M., Proepper, C., Brunner, C., Katz, S., Pusapati, G. V., Bullinger, L., Franz, W., Koehntop, R., Giehl, K., Spyrantis, A., Wittekindt, O., Lin, Q., Zenke, M., Fleischmann, B. K., Wartenberg, M., Wobus, A. M., Boeckers, T. M., Liebau, S. 2010; 122 (18): 1823-?


    Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage.We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone-induced effects.SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.

    View details for DOI 10.1161/CIRCULATIONAHA.110.971721

    View details for Web of Science ID 000283670800013

    View details for PubMedID 20956206

  • Protein kinase D2 is a crucial regulator of tumour cell-endothelial cell communication in gastrointestinal tumours GUT Azoitei, N., Pusapati, G. V., Kleger, A., Moeller, P., Kuefer, R., Genze, F., Wagner, M., Van Lint, J., Carmeliet, P., Adler, G., Seufferlein, T. 2010; 59 (10): 1316-1330


    Tumour angiogenesis is crucially dependent on the communication between the tumour and the associated endothelium. Protein kinase D (PKD) isoenzymes mediate vascular endothelial growth factor-A (VEGF-A) induced endothelial cell proliferation and migration and are also highly expressed in various tumours.To examine the role of PKDs for tumour proliferation and angiogenesis selectively in pancreatic and gastric tumours and in tumour-associated endothelium in vitro and in vivo.PKD2 expression in human tumours was determined by immunohistochemistry. The effect of PKD2 depletion in endothelial cells by siRNAs was examined in sprouting assays, the chorioallantois model (CAM) and tumour xenografts. In murine endothelium in vivo PKD2 was knocked-down by splice switching oligonucleotides. Human PKD2 was depleted in xenografts by siRNAs and PKD2-miRs. PKD2 activation by hypoxia and its role for hypoxia-induced NR4/TR3- and VEGF-A promoter activity, expression and secretion was investigated in cell lines.PKD2 is expressed in gastrointestinal tumours and in the tumour-associated endothelium. Tumour growth and angiogenesis in the CAM and in tumour xenografts require PKD expression in endothelial cells. Conversely, hypoxia activates PKD2 in pancreatic cancer cells and PKD2 was identified as the major mediator of hypoxia-stimulated VEGF-A promoter activity, expression and secretion in tumour cells. PKD2 depletion in pancreatic tumours inhibited tumour-driven blood vessel formation and tumour growth in the CAM and in orthotopic pancreatic cancer xenografts.PKD2 regulates hypoxia-induced VEGF-A expression/secretion by tumour cells and VEGF-A stimulated blood vessel formation. PKD2 is a novel, essential mediator of tumour cell-endothelial cell communication and a promising therapeutic target to inhibit angiogenesis in gastrointestinal cancers.

    View details for DOI 10.1136/gut.2009.206813

    View details for Web of Science ID 000282661300006

    View details for PubMedID 20732914

  • Role of the Second Cysteine-rich Domain and Pro275 in Protein Kinase D2 Interaction with ADP-Ribosylation Factor 1, Trans-Golgi Network Recruitment, and Protein Transport MOLECULAR BIOLOGY OF THE CELL Pusapati, G. V., Krndija, D., Armacki, M., von Wichert, G., Von Blume, J., Malhotra, V., Adler, G., Seufferlein, T. 2010; 21 (6): 1011-1022


    Protein kinase D (PKD) isoenzymes regulate the formation of transport carriers from the trans-Golgi network (TGN) that are en route to the plasma membrane. The PKD C1a domain is required for the localization of PKDs at the TGN. However, the precise mechanism of how PKDs are recruited to the TGN is still elusive. Here, we report that ADP-ribosylation factor (ARF1), a small GTPase of the Ras superfamily and a key regulator of secretory traffic, specifically interacts with PKD isoenzymes. ARF1, but not ARF6, binds directly to the second cysteine-rich domain (C1b) of PKD2, and precisely to Pro275 within this domain. Pro275 in PKD2 is not only crucial for the PKD2-ARF1 interaction but also for PKD2 recruitment to and PKD2 function at the TGN, namely, protein transport to the plasma membrane. Our data suggest a novel model in which ARF1 recruits PKD2 to the TGN by binding to Pro275 in its C1b domain followed by anchoring of PKD2 in the TGN membranes via binding of its C1a domain to diacylglycerol. Both processes are critical for PKD2-mediated protein transport.

    View details for DOI 10.1091/mbc.E09-09-0814

    View details for Web of Science ID 000275433500016

    View details for PubMedID 20089835