07-472 Sigma-AldrichAnti-PTPα Antibody

Cell-cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell-cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha-Src family kinase-Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin-based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.

Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY) proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC). We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKCδ), CUB-domain-containing protein 1 (CDCP1), Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2), and receptor protein-tyrosine phosphatase alpha (RPTPα). The pY313 site PKCδ was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKCδ pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKCδ pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers.

Controlled production of reactive oxygen species leads to reversible oxidation of protein tyrosine phosphatases (PTPs) and has emerged as an important tier of regulation over phosphorylation-dependent signal transduction. We present a modified cysteinyl-labeling assay that detects reversible oxidation of members of each of the different PTP subclasses. Here, we describe the methods for enriching reversibly oxidized PTPs from complex protein extracts, illustrating the procedure in IMR90 fibroblasts.

During mitosis, the catalytic activity of protein-tyrosine phosphatase (PTP) alpha is enhanced, and its inhibitory binding to Grb2, which specifically blocks Src dephosphorylation, is decreased. These effects act synergistically to activate Src in mitosis. We show here that these effects are abrogated by mutation of Ser180 and/or Ser204, the sites of protein kinase C-mediated phosphorylation within PTPalpha. Moreover, either a Ser-to-Ala substitution or serine dephosphorylation specifically eliminated the ability of PTPalpha to dephosphorylate and activate Src even during interphase. This explains why the substitutions eliminated PTPalpha transforming activity, even though PTPalpha interphase dephosphorylation of nonspecific substrates was only slightly decreased. This occurred without change in the phosphorylation of PTPalpha at Tyr789, which is required for "phosphotyrosine displacement" during Src dephosphorylation. Thus, in addition to increasing PTPalpha nonspecific catalytic activity, Ser180 and Ser204 phosphorylation (along with Tyr789 phosphorylation) regulates PTPalpha substrate specificity. This involves serine phosphorylation-dependent differential modulation of the affinity of Tyr(P)789 for the Src and Grb2 SH2 domains. The results suggest that protein kinase C may participate in the mitotic activation of PTPalpha and Src and that there are intramolecular interactions between the PTPalpha C-terminal and membrane-proximal regions that are regulated, at least in part, by serine phosphorylation.

We show that, dependent on serine hyperphosphorylation, protein tyrosine phosphatase alpha (PTPalpha) is activated by two different mechanisms during mitosis: its specific activity increases and its inhibitory binding to Grb2 decreases. The latter effect probably abates Grb2 inhibition of the phosphotyrosine displacement process that is required specifically for Src dephosphorylation and causes a mitotic increase in transient PTPalpha-Src binding. Thus, part of the increased protein tyrosine phosphatase activity may be specific for Src family members. These effects cease along with Src activation when cells exit mitosis. Src is not activated in mitosis in PTPalpha-knockout cells, indicating a unique mitotic role for this phosphatase. The activation of PTPalpha, combined with the effects of mitotic Cdc2-mediated phosphorylations of Src, quantitatively accounts for the mitotic activation of Src, indicating that PTPalpha is the membrane-bound, serine phosphorylation-activated, protein tyrosine phosphatase that activates Src during mitosis.

Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity of a pathway, can be controlled by multiple tyrosine phosphatases, attesting to the intricate integration of these enzymes in cellular regulation. Lastly, we are starting to appreciate the roles of intracellular topology, tyrosine phosphorylation and oligomerization among the many mechanisms regulating tyrosine phosphatase activity.