Biology subjects

Torres Robles, J.

Publications and source records attributed to Torres Robles, J..

3 recordsLinked to original sources

bioRxiv2022

Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to a wide array of stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling specific inputs to appropriate cellular responses. One way that MAPKs achieve specificity is through transient interactions with docking sites: short linear motifs found in MAPK substrates, regulators, and scaffolds. Docking sites bind to a conserved groove located in the catalytic domain of all MAPKs including the ERK and p38 subfamilies, but how specificity is achieved remains unresolved. To understand the basis of docking selectivity for these two subfamilies, we screened a library of thousands of human proteome-derived sequences for docking to ERK2 and p38. We discovered a large number of sequences that bound specifically to only one MAPK or promiscuously to both, and that selective and non-selective interactors conformed to distinct sequence motifs. In particular, selective binding to p38 correlated with higher net charge in the docking site, and this phenomenon was driven by enrichment for Lys residues. A pair of acidic residues unique to the docking groove of p38 mediated selectivity for Lys-rich basic motifs. Finally, we validated a set of full-length proteins harboring docking sites selected as hits in our screens to be authentic MAPK interactors and identified ChREBP and TACC1 as cellular MAPK substrates. This study identifies distinguishing features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.

cell biology↗

bioRxiv2022

A global atlas of substrate specificities for the human serine/threonine kinome

Protein phosphorylation is one of the most widespread post-translational modifications in biology. With the advent of mass spectrometry-based phosphoproteomics, more than 200,000 sites of serine and threonine phosphorylation have been reported, of which several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein Ser/Thr kinases encoded in the human genome is responsible. Here, we utilize synthetic peptide libraries to profile the substrate sequence specificity of nearly every functional human Ser/Thr kinase. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. Our kinome-wide dataset was used to computationally annotate and identify the most likely protein kinases for every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites where the protein kinases involved have been previously identified, our predictions were in excellent agreement. When this approach was applied to examine the signaling 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 full extent of substrate specificity of the human Ser/Thr kinome, illuminate cellular signaling responses, and provide a rich resource to link unannotated phosphorylation events to biological pathways.

systems biology↗

bioRxiv2021

Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors

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 p38. We subsequently screened a library consisting of ~12,000 sequences from the human proteome, revealing a large number of MAPK-selective interactors, including many not conforming to previously defined docking motifs. Analysis of p38/JNK1 exchange mutants identified specific docking groove residues mediating selective binding. Finally, we verified that docking sequences identified in the screen could function in substrate recruitment in vitro and in cultured cells. Collectively, these studies establish an approach for characterization of MAPK docking sequences and provide a resource for future investigation of signaling downstream of p38 and JNK MAP kinases.

cell biology↗