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The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition., (© 2014 John Wiley & Sons A/S.)

Protein arginine methyltransferase 1 (PRMT1)-dependent methylation contributes to the onset and progression of numerous diseases (e.g., cancer, heart disease, ALS); however, the regulatory mechanisms that control PRMT1 activity are relatively unexplored. We therefore set out to decipher how phosphorylation regulates PRMT1 activity. Curated mass spectrometry data identified Tyr291, a residue adjacent to the conserved THW loop, as being phosphorylated. Natural and unnatural amino acid mutagenesis, including the incorporation of p-carboxymethyl-l-phenylalanine (pCmF) as a phosphotyrosine mimic, were used to show that Tyr291 phosphorylation alters the substrate specificity of PRMT1. Additionally, p-benzoyl-l-phenylalanine (pBpF) was incorporated at the Tyr291 position, and cross-linking experiments with K562 cell extracts identified several proteins (e.g., hnRNPA1 and hnRNP H3) that bind specifically to this site. Moreover, we also demonstrate that Tyr291 phosphorylation impairs PRMT1's ability to bind and methylate both proteins. In total, these studies demonstrate that Tyr291 phosphorylation alters both PRMT1 substrate specificity and protein-protein interactions.

Protein arginine methyltransferase (PRMT) 8 is unique among the PRMTs, as it has a highly restricted tissue expression pattern and an N terminus that contains two automethylation sites and a myristoylation site. PRMTs catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) to a peptidylarginine on a protein substrate. Currently, the physiological roles, regulation, and cellular substrates of PRMT8 are poorly understood. However, a thorough understanding of PRMT8 kinetics should provide insights into each of these areas, thereby enhancing our understanding of this unique enzyme. In this study, we determined how automethylation regulates the enzymatic activity of PRMT8. We found that preventing automethylation with lysine mutations (preserving the positive charge of the residue) increased the turnover rate and decreased the Km of AdoMet but did not affect the Km of the protein substrate. In contrast, mimicking automethylation with phenylalanine (i.e. mimicking the increased hydrophobicity) decreased the turnover rate. The inhibitory effect of the PRMT8 N terminus could be transferred to PRMT1 by creating a chimeric protein containing the N terminus of PRMT8 fused to PRMT1. Thus, automethylation of the N terminus likely regulates PRMT8 activity by decreasing the affinity of the enzyme for AdoMet.

The best characterized examples of crosstalk between two or more different post-translational modifications (PTMs) occur with respect to histones. These examples demonstrate the critical roles that crosstalk plays in regulating cell signaling pathways. Recently, however, non-histone crosstalk has been observed between serine/threonine phosphorylation and the modification of arginine and lysine residues within kinase consensus sequences. Interestingly, many kinase consensus sequences contain critical arginine/lysine residues surrounding the substrate serine/threonine residue. Therefore, we hypothesize that non-histone crosstalk between serine/threonine phosphorylation and arginine/lysine modifications is a global mechanism for the modulation of cellular signaling. In this review, we discuss several recent examples of non-histone kinase consensus sequence crosstalk, as well as provide the biophysical basis for these observations. In addition, we predict likely examples of crosstalk between protein arginine methyltransferase 1 (PRMT1) and Akt and discuss the future implications of these findings.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidinium group of arginine residues in a number of important cell signaling proteins. PRMT1 is the founding member of this family, and its activity appears to be dysregulated in heart disease and cancer. To begin to characterize the catalytic mechanism of this isozyme, we assessed the effects of mutating a number of highly conserved active site residues (i.e., Y39, R54, E100, E144, E153, M155, and H293), which are believed to play key roles in SAM recognition, substrate binding, and catalysis. The results of these studies, as well as pH-rate studies, and the determination of solvent isotope effects (SIEs) indicate that M155 plays a critical role in both SAM binding and the processivity of the reaction but is not responsible for the regiospecific formation of asymmetrically dimethylated arginine (ADMA). Additionally, mutagenesis studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general base. Furthermore, the lack of a normal SIE with either the wild type or catalytically impaired mutants suggests that general acid/base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1-catalyzed reaction is primarily driven by bringing the substrate guanidinium into the proximity of the S-methyl group of SAM and that the prior deprotonation of the substrate guanidinium is not required for methyl transfer.

The dysregulation of protein arginine methyltransferases (PRMTs) is implicated in a wide variety of disease states. Here we report the design, synthesis, and screening of a combinatorial peptide library used to characterize the substrate specificity of PRMT1. The information gained from this approach was used to develop a PRMT1 inhibitor with enhanced selectivity.