Paralog-Specific Patterns of Structural Disorder and Phosphorylation in the Vertebrate SH3–SH2–Tyrosine Kinase Protein Family

One of the largest multigene families in Metazoa are the tyrosine kinases (TKs). These are important multifunctional proteins that have evolved as dynamic switches that perform tyrosine phosphorylation and other noncatalytic activities regulated by various allosteric mechanisms. TKs interact with each other and with other molecules, ultimately activating and inhibiting different signaling pathways. TKs are implicated in cancer and almost 30 FDA-approved TK inhibitors are available. However, specific binding is a challenge when targeting an active site that has been conserved in multiple protein paralogs for millions of years. A cassette domain (CD) containing SH3–SH2–Tyrosine Kinase domains reoccurs in vertebrate nonreceptor TKs. Although part of the CD function is shared between TKs, it also presents TK specific features. Here, the evolutionary dynamics of sequence, structure, and phosphorylation across the CD in 17 TK paralogs have been investigated in a large-scale study. We establish that TKs often have ortholog-specific structural disorder and phosphorylation patterns, while secondary structure elements, as expected, are highly conserved. Further, domain-specific differences are at play. Notably, we found the catalytic domain to fluctuate more in certain secondary structure elements than the regulatory domains. By elucidating how different properties evolve after gene duplications and which properties are specifically conserved within orthologs, the mechanistic understanding of protein evolution is enriched and regions supposedly critical for functional divergence across paralogs are highlighted

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition

Inherited duplications of PPP2R3B predispose to nevi and melanoma via a C21orf91-driven proliferative phenotype

Purpose Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. Methods Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. Results We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. Conclusion This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching

The effect of the N-terminal HEAT repeats of the A-subunit on methylation.

<p>(<b>A</b>) Schematic representation of internal truncations of the N-terminal HEAT repeats to reduce the length of the A-subunit N-terminal structure. (<b>B</b>) Normalized concentrations of PP2Ac assembled with full-length or truncated A-subunits used for kinetic analysis of PP2A methylation in (<b>C</b>). (<b>C</b>) Kinetics of methylation of PP2A core enzymes with varied length of the A-subunit. Results from 6–7 independent experiments were summarized and the average K_m ± std. dev. was calculated and shown in the table on the right. (<b>D</b>) Correlation between the number of deleted N-terminal HEAT repeats and the K_m of methylation of PP2A core enzyme by LCMT-1. Statistical test of this correlation was performed and the P value was calculated and shown. (<b>E</b>) Structure of the core enzyme portion of PP2A holoenzyme (PDB ID: 2NPP). The A-subunit, PP2Ac, and PP2Ac-tail are colored green, blue, and magenta, respectively. The positions of the HEAT-repeats where truncations were made are indicated by numbers.</p

Electrostatic interactions between the A-subunit and LCMT-1.

<p>(<b>A</b>) Structural model of the PP2A core enzyme-LCMT-1 complex built from the structure of the PP2Ac-LCMT-1 complex and the A-subunit with morphed conformation. The electrostatic potential for the A-subunit (left panel) and LCMT-1 (right panel) is shown. The corresponding regions on the A-subunit and LCMT-1 for potential electrostatic interactions are circled (i and ii). The A-subunit residues that directly contact LCMT-1 in the model, Arg183 and Arg258, are shown. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086955#pone.0086955.s003" target="_blank">Movie S1</a>. (<b>B</b>) The effect of the A-subunit mutations to Arg183 and Arg258 on methylation of the PP2A core enzyme. Results from 5–7 independent experiments were summarized and the average K_m ± std. dev. and the P value were calculated and shown. <b>(C)</b> FRET assay measured changes in the distance between the N- and C-termini of the A-subunit in the core enzyme prior to and after addition of an excess molar amount of LCMT-1 or a stoichiometric amount of PR70 (108–575). Representative results were shown with mean ± SEM calculated from triplicate.</p

Mechanisms of the Scaffold Subunit in Facilitating Protein Phosphatase 2A Methylation

<div><p>The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A–C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.</p></div

Predicting kinase inhibitors using bioactivity matrix derived informer sets

10.1371/journal.pcbi.1006813PLoS Computational Biology158e100681