Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

The protein tyrosine phosphatase nonreceptor type 12 (PTPN12) is a multifunctional protein and has elicited much research attention because its decreased protein level has been associated with poor prognosis of several types of cancers. Recently, we have solved the crystal structure of the phosphatase domain of PTPN12, which disclosed a specific PTPN12-insert-loop harboring a cyclin-dependent kinase 2 (CDK2) phosphorylation site. However, the functional significance of this phosphorylation is undefined. In the present study, we found that S19 site phosphorylation of PTPN12 by CDK2 discharged its antitumor activity by down-regulation of its inhibitory role in cell migration, but not affecting its other regulatory functions. Phosphorylation of PTPN12 at the S19 site changed its substrate interface, and by doing so, selectively decreased its activity toward the human epidermal growth factor receptor 2 (HER2)- pY1196 site, but not other HER2 phosphorylation sites or other known PTPN12 substrates. A further in-depth mechanism study revealed that the phosphorylation of PTPN12 by CDK2 impaired recruitment of the serine/threonine-protein kinase 1 (PAK1) to HER2, resulted in the blockade of the HER2-pY1196-PAK1-T423 signaling pathway, thus increased tumor cell motility. Taken together, our results identified a new phosphorylation-based substrate recognition mechanism of PTPN12 by CDK2, which orchestrated signaling crosstalk between the oncogenic CDK2 and HER2 pathways. The newly identified governing mechanism of the substrate selectivity of a particular phosphatase was previously unappreciated and exemplifies how a phospho-network is precisely controlled in different cellular contexts.-Li, H., Yang, D., Ning, S., Xu, Y., Yang, F., Yin, R., Feng, T., Han, S., Guo, L., Zhang, P., Qu, W., Guo, R., Song, C., Xiao, P., Zhou, C., Xu, Z., Sun, J.-P., Yu, X. Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

Structure and Molecular Dynamics Simulations of Protein Tyrosine Phosphatase Non-Receptor 12 Provide Insights into the Catalytic Mechanism of the Enzyme.

Protein tyrosine phosphatase non-receptor 12 (PTPN12) is an important protein tyrosine phosphatase involved in regulating cell adhesion and migration as well as tumorigenesis. Here, we solved a crystal structure of the native PTPN12 catalytic domain with the catalytic cysteine (residue 231) in dual conformation (phosphorylated and unphosphorylated). Combined with molecular dynamics simulation data, we concluded that those two conformations represent different states of the protein which are realized during the dephosphorylation reaction. Together with docking and mutagenesis data, our results provide a molecular basis for understanding the catalytic mechanism of PTPN12 and its role in tumorigenesis.

Crystal Structure and Substrate Specificity of PTPN12.

PTPN12 is an important tumor suppressor that plays critical roles in various physiological processes. However, the molecular basis underlying the substrate specificity of PTPN12 remains uncertain. Here, enzymological and crystallographic studies have enabled us to identify two distinct structural features that are crucial determinants of PTPN12 substrate specificity: the pY+1 site binding pocket and specific basic charged residues along its surface loops. Key structurally plastic regions and specific residues in PTPN12 enabled recognition of different HER2 phosphorylation sites and regulated specific PTPN12 functions. In addition, the structure of PTPN12 revealed a CDK2 phosphorylation site in a specific PTPN12 loop. Taken together, our results not only provide the working mechanisms of PTPN12 for desphosphorylation of its substrates but will also help in designing specific inhibitors of PTPN12.

Identification of Novel Candidate Genes for Early-Onset Colorectal Cancer Susceptibility.

Approximately 25-30% of colorectal cancer (CRC) cases are expected to result from a genetic predisposition, but in only 5-10% of these cases highly penetrant germline mutations are found. The remaining CRC heritability is still unexplained, and may be caused by a hitherto-undefined set of rare variants with a moderately penetrant risk. Here we aimed to identify novel risk factors for early-onset CRC using whole-exome sequencing, which was performed on a cohort of CRC individuals (n = 55) with a disease onset before 45 years of age. We searched for genes that were recurrently affected by rare variants (minor allele frequency ≤ 0.001) with potentially damaging effects and, subsequently, re-sequenced the candidate genes in a replication cohort of 174 early-onset or familial CRC individuals. Two functionally relevant genes with low frequency variants with potentially damaging effects, PTPN12 and LRP6, were found in at least three individuals. The protein tyrosine phosphatase PTP-PEST, encoded by PTPN12, is a regulator of cell motility and LRP6 is a component of the WNT-FZD-LRP5-LRP6 complex that triggers WNT signaling. All variants in LRP6 were identified in individuals with an extremely early-onset of the disease (≤30 years of age), and two of the three variants showed increased WNT signaling activity in vitro. In conclusion, we present PTPN12 and LRP6 as novel candidates contributing to the heterogeneous susceptibility to CRC.

Mosaic partial deletion of PTPN12 in a child with interrupted aortic arch type A.

Congenital heart malformations, including those of the great vessels, are among the most common human birth defects. The goal of this study was to identify the significance of a de novo mosaic PTPN12 partial deletion identified in a newborn with an interrupted aortic arch type A, ventricular septal defect, and pyloric stenosis. PTPN12, a downstream target of the RAS pathway, has a known role in endothelial cell adhesion and migration. Neither genetic nor genomic variants in PTPN12 have been described in a human patient; therefore, we evaluated the effect of ptpn12 in a mouse conditional knockout and zebrafish knockdown model to determine the significance of a loss in gene expression. Observed loss of ptpn12 expression in zebrafish resulted in abnormal branchial arch and tail vasculature patterns, with reduced blood flow throughout the animal. This phenotype was supported by anomalous vasculature in a conditional Ptpn12 mouse knockout. Given the novel co-occurrence of interrupted aortic arch type A, ventricular septal defect, and partial deletion of PTPN12 in the patient, as well as vascular phenotypes in Ptpn12 mouse and ptpn12 zebrafish models, it is likely that PTPN12 has a significant role in cardiovascular development and vessel formation during human embryonic development. Furthermore, the partial deletion of PTPN12 lead to interrupted aortic arch type A in this child and may represent a novel condition caused by a null mutation in the RAS pathway.

Profiling the substrate specificity of protein kinases by on-bead screening of peptide libraries.

A robust, high-throughput method has been developed to screen one-bead-one-compound peptide libraries to systematically profile the sequence specificity of protein kinases. Its ability to provide individual sequences of the preferred substrates permits the identification of sequence contextual effects and nonpermissive residues. Application of the library method to kinases Pim1, MKK6, and Csk revealed that Pim1 and Csk are highly active toward peptide substrates and recognize specific sequence motifs, whereas MKK6 has little activity or sequence selectivity against peptide substrates. Pim1 recognizes peptide substrates of the consensus RXR(H/R)X(S/T); it accepts essentially any amino acid at the S/T-2 and S/T+1 positions, but strongly disfavors acidic residues (Asp or Glu) at the S/T-2 position and a proline residue at the S/T+1 position. The selected Csk substrates show strong sequence covariance and fall into two classes with the consensus sequences of (D/E)EPIYϕXϕ and (D/E)(E/D)S(E/D/I)YϕXϕ (where X is any amino acid and ϕ is a hydrophobic amino acid). Database searches and in vitro kinase assays identified phosphatase PTP-PEST as a Pim1 substrate and phosphatase SHP-1 as a potential Csk substrate. Our results demonstrate that the sequence specificity of protein kinases is defined not only by favorable interactions between permissive residue(s) on the substrate and their cognate binding site(s) on the kinase but also by repulsive interactions between the kinase and nonpermissive residue(s).

Protein tyrosine phosphatase with proline-glutamine-serine-threonine-rich motifs negatively regulates TLR-triggered innate responses by selectively inhibiting IκB kinase ß/NF-κB activation.

TLRs are essential for sensing the invading pathogens and initiating protective immune responses. However, aberrant activation of TLR-triggered inflammatory innate responses leads to the inflammatory disorders and autoimmune diseases. The molecular mechanisms that fine-tune TLR responses remain to be fully elucidated. Protein tyrosine phosphatase with proline-glutamine-serine-threonine-rich motifs (PTP-PEST) has been shown to be important in cell adhesion, migration, and also T cell and B cell activation. However, the roles of PTP-PEST in TLR-triggered immune response remain unclear. In this study, we report that PTP-PEST expression was upregulated in macrophages by TLR ligands. PTP-PEST inhibited TNF-α, IL-6, and IFN-ß production in macrophages triggered by TLR3, TLR4, and TLR9. Overexpression of catalytically inactive mutants of PTP-PEST abolished the inhibitory effects, indicating that PTP-PEST inhibits TLR response in a phosphatase-dependent manner. Accordingly, PTP-PEST knockdown increased TLR3, -4, and -9-triggered proinflammatory cytokine and type I IFN production. PTP-PEST selectively inhibited TLR-induced NF-κB activation, whereas it had no substantial effect on MAPK and IFN regulatory factor 3 activation. Moreover, PTP-PEST directly interacted with IκB kinase ß (IKKß) then inhibited IKKß phosphorylation at Ser(177/181) and Tyr(188/199), and subsequently suppressed IKKß activation and kinase activity as well as downstream NF-κB activation, resulting in suppression of the TLR-triggered innate immune response. Thus, PTP-PEST functions as a feedback-negative regulator of TLR-triggered innate immune responses by selectively impairing IKKß/NF-κB activation.

Chemical rescue of protein tyrosine phosphatase activity.

We report that the activity of a rationally engineered protein tyrosine phosphatase (PTP) mutant can be fully rescued by the addition of the biarsenical fluorescein derivative FlAsH, a compound that does not affect the activity of wild-type PTPs.

Gold(I) phosphine mediated selective inhibition of lymphoid tyrosine phosphatase.

Selective protein tyrosine phosphatase (PTP) inhibition is often difficult to achieve owing to the high degree of similarity of the catalytic domains of this family of enzymes. Selective inhibitors of the lymphoid specific tyrosine phosphatase, LYP, are of great interest due to the involvement of LYP in several autoimmune disorders. This manuscript describes a study into the mechanistic details of selective LYP inhibition by a Au(I)-phosphine complex. The complex, [Au((CH(2)CH(2)CN)(2)PPh)Cl], selectively inhibits LYP activity both in vitro and in cells, but does not inhibit other T-cell derived PTPs including the highly homologous PTP-PEST. The mode of inhibition was probed by investigating inhibition of LYP, the LYP mutant C129/231S, and PTP-PEST. Inhibition of LYP and PTP-PEST was competitive, while the LYP double mutant appeared mixed. Wild-type LYP was inhibited more potently than LYP C129/231S, indicating an important role for at least one of these residues in Au(I) binding. Coordination of Au(I) by both the active site cysteine residue as well as either Cys129 or 231 is suggested as a potential mechanism for LYP selective inhibition.

Protein phosphatase 1alpha associates with protein tyrosine phosphatase-PEST inducing dephosphorylation of phospho-serine 39.

Protein tyrosine phosphatase (PTP)-PEST is expressed in a wide variety of several cell types and is an efficient regulator of cell adhesion, spreading and migration. PTP-PEST-associating molecules are important in elucidating the function of PTP-PEST. Herein, we have identified protein phosphatase 1alpha (PP1alpha) as a novel PTP-PEST binding protein, and then we aimed to determine how PP1alpha contributes to the phosphorylation at Ser39 of PTP-PEST, whose phosphorylation suppresses PTP-PEST enzymatic activity. The HEK 293 cells overexpressing exogenous PTP-PEST were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and the phosphorylation of PTP-PEST at Ser39 was evaluated using an anti-phospho-Ser39 PTP-PEST specific antibody (anti-pS39-PEST Ab). It was demonstrated that the phosphorylation at Ser39 detected by anti-pS39-PEST Ab was dependent on TPA treatment and a significant inverse correlation between the PTP activity of PTP-PEST and anti-pS39-PEST Ab-immunoreactive band intensity. The phosphorylation of Ser39 was suppressed by co-transfection of a plasmid encoding wild-type PP1alpha, but not by that of the dominant-negative PP1alpha mutant. Furthermore, TPA-induced phosphorylation could take place in PTP-PEST catalytic domain, but the phosphorylation of PTP-PEST catalytic domain could not be abrogated by co-transfection of a plasmid expressing wild-type PP1alpha. In conclusion, PP1alpha associates with the non-catalytic domain of PTP-PEST and regulates PTP activity via dephosphorylation of phospho-Ser39.