Jaylissa Torres Robles

All Publications

• Cancer hotspot mutations rewire ERK2 specificity by selective exclusion of docking interactions JOURNAL OF BIOLOGICAL CHEMISTRY Robles, J., Stiegler, A. L., Boggon, T. J., Turk, B. E. 2025; 301 (4): 108348

  Abstract

  The protein kinase ERK2 is recurrently mutated in human squamous cell carcinomas and other tumors. ERK2 mutations cluster in an essential docking recruitment site that interacts with short linear motifs found within intrinsically disordered regions of ERK substrates and regulators. Cancer-associated mutations do not disrupt ERK2 docking interactions altogether but selectively inhibit some interactions while sparing others. However, the full scope of disrupted or maintained interactions remains unknown, limiting our understanding of how these mutations contribute to cancer. We recently defined the docking interactome of wild-type ERK2 by screening a yeast two-hybrid library of proteomic short linear motifs. Here, we apply this approach to the two most recurrent cancer-associated mutants. We find that most sequences binding to WT ERK2 also interact with both mutant forms. Analysis of differentially interacting sequences revealed that ERK2 mutants selectively lose the ability to bind sequences conforming to a specific motif. We solved the co-crystal structure of ERK2 in complex with a peptide fragment of ISG20, a screening hit that binds exclusively to the WT kinase. This structure demonstrated the mechanism by which cancer hotspot mutations at Glu81, Arg135, Asp321, and Glu322 selectively impact peptide binding. Finally, we found that cancer-associated ERK2 mutations had decreased activity in phosphorylating GEF-H1/ARHGEF2, a known ERK substrate harboring a WT-selective docking motif. Collectively, our studies provide a structural rationale for how a broad set of interactions are disrupted by ERK2 hotspot mutations, suggesting mechanisms for pathway rewiring in cancers harboring these mutations.

  View details for DOI 10.1016/j.jbc.2025.108348

  View details for Web of Science ID 001468214700001

  View details for PubMedID 40015635

  View details for PubMedCentralID PMC11982978

• Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Robles, J., Lou, H., Shi, G., Pan, P., Turk, B. E. 2023; 120 (48): e2316599120

  Abstract

  Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites-short linear motifs found in MAPK substrates, regulators, and scaffolds-can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.

  View details for DOI 10.1073/pnas.2316599120

  View details for Web of Science ID 001157391900011

  View details for PubMedID 37988460

  View details for PubMedCentralID PMC10691213

• An atlas of substrate specificities for the human serine/threonine kinome NATURE Johnson, J. L., Yaron, T. M., Huntsman, E. M., Kerelsky, A., Song, J., Regev, A., Lin, T., Liberatore, K., Cizin, D. M., Cohen, B. M., Vasan, N., Ma, Y., Krismer, K., Robles, J., van de Kooij, B., van Vlimmeren, A. E., Andree-Busch, N., Kaeufer, N. F., Dorovkov, M., Ryazanov, A. G., Takagi, Y., Kastenhuber, E. R., Goncalves, M. D., Hopkins, B. D., Elemento, O., Taatjes, D. J., Maucuer, A., Yamashita, A., Degterev, A., Uduman, M., Lu, J., Landry, S. D., Zhang, B., Cossentino, I., Linding, R., Blenis, J., Hornbeck, P., Turk, B. E., Yaffe, M. B., Cantley, L. C. 2023; 613 (7945): 759-+

  Abstract

  Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

  View details for DOI 10.1038/s41586-022-05575-3

  View details for Web of Science ID 000913868600004

  View details for PubMedID 36631611

  View details for PubMedCentralID PMC9876800

• Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors SCIENCE SIGNALING Shi, G., Song, C., Robles, J., Salichos, L., Lou, H., Lam, T. T., Gerstein, M., Turk, B. E. 2023; 16 (767): eabm5518

  Abstract

  Essential functions of mitogen-activated protein kinases (MAPKs) depend on their capacity to selectively phosphorylate a limited repertoire of substrates. MAPKs harbor a conserved groove located outside of the catalytic cleft that binds to short linear sequence motifs found in substrates and regulators. However, the weak and transient nature of these "docking" interactions poses a challenge to defining MAPK interactomes and associated sequence motifs. Here, we describe a yeast-based genetic screening pipeline to evaluate large collections of MAPK docking sequences in parallel. Using this platform, we analyzed a combinatorial library based on the docking sequences from the MAPK kinases MKK6 and MKK7, defining features critical for binding to the stress-activated MAPKs JNK1 and p38α. Our screen of a library consisting of ~12,000 sequences from the human proteome revealed multiple MAPK-selective interactors, including many that did not conform to previously defined docking motifs. Analysis of p38α/JNK1 exchange mutants identified specific docking groove residues that mediate selective binding. Last, we verified that docking sequences identified in the screen functioned in substrate recruitment in vitro and in cultured cells. Together, these studies establish an approach to characterize MAPK docking sequences and provide a resource for future investigation of signaling downstream of p38 and JNK.

  View details for DOI 10.1126/scisignal.abm5518

  View details for Web of Science ID 000933631100002

  View details for PubMedID 36626580

  View details for PubMedCentralID PMC9995140