The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain.

Mutations in the tyrosine phosphatase SHP2 are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting auto-inhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that, while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8-10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.

An atlas of substrate specificities for the human serine/threonine kinome.

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.

MDM2, MDMX, and p73 regulate cell-cycle progression in the absence of wild-type p53.

The p53 tumor suppressor protein, known to be critically important in several processes including cell-cycle arrest and apoptosis, is highly regulated by multiple mechanisms, most certifiably the Murine Double Minute 2-Murine Double Minute X (MDM2-MDMX) heterodimer. The role of MDM2-MDMX in cell-cycle regulation through inhibition of p53 has been well established. Here we report that in cells either lacking p53 or expressing certain tumor-derived mutant forms of p53, loss of endogenous MDM2 or MDMX, or inhibition of E3 ligase activity of the heterocomplex, causes cell-cycle arrest. This arrest is correlated with a reduction in E2F1, E2F3, and p73 levels. Remarkably, direct ablation of endogenous p73 produces a similar effect on the cell cycle and the expression of certain E2F family members at both protein and messenger RNA levels. These data suggest that MDM2 and MDMX, working at least in part as a heterocomplex, may play a p53-independent role in maintaining cell-cycle progression by promoting the activity of E2F family members as well as p73, making them a potential target of interest in cancers lacking wild-type p53.

Timing the origin of eukaryotic cellular complexity with ancient duplications.

Eukaryogenesis is one of the most enigmatic evolutionary transitions, during which simple prokaryotic cells gave rise to complex eukaryotic cells. While evolutionary intermediates are lacking, gene duplications provide information on the order of events by which eukaryotes originated. Here we use a phylogenomics approach to reconstruct successive steps during eukaryogenesis. We find that gene duplications roughly doubled the proto-eukaryotic gene repertoire, with families inherited from the Asgard archaea-related host being duplicated most. By relatively timing events using phylogenetic distances, we inferred that duplications in cytoskeletal and membrane-trafficking families were among the earliest events, whereas most other families expanded predominantly after mitochondrial endosymbiosis. Altogether, we infer that the host that engulfed the proto-mitochondrion had some eukaryote-like complexity, which drastically increased upon mitochondrial acquisition. This scenario bridges the signs of complexity observed in Asgard archaeal genomes to the proposed role of mitochondria in triggering eukaryogenesis.

Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs.

Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the -3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif amplifiers of Plk1 signaling.