Abstract
Tensin1 is the archetype of a family of focal adhesion proteins. Tensin1 has a phosphotyrosine binding domain that binds the cytoplasmic tail of β-integrin, a Src homology 2 domain that binds focal adhesion kinase, p130Cas, and the RhoGAP called deleted in liver cancer-1, a phosphatase and tensin homology domain that binds protein phosphatase-1α and other regions that bind F-actin. The association between tensin1 and these partners affects cell polarization, migration, and invasion. In this study we analyzed the phosphorylation of human S-tag-tensin1 expressed in HEK293 cells by mass spectrometry. Peptides covering >90% of the sequence initially revealed 50 phosphorylated serine/phosphorylated threonine (pSer/pThr) but no phosphorylated tyrosine (pTyr) sites. Addition of peroxyvanadate to cells to inhibit protein tyrosine phosphatases exposed 10 pTyr sites and addition of calyculin A to cells to inhibit protein phosphatases type 1 and 2A gave a total of 62 pSer/pThr sites. We also characterized two sites modified by O-linked N-acetylglucosamine. Tensin1 F302A, which does not bind protein phosphatase-1, showed > twofold enhanced phosphorylation of seven sites. The majority of pSer/pThr have adjacent proline (Pro) residues and we show endogenous p38 mitogen activated protein kinase (MAPK) associated with and phosphorylated tensin1 in an in vitro kinase assay. Recombinant p38α MAPK also phosphorylated S-tag-tensin1, resulting in decreased binding with deleted in liver cancer-1. Activation of p38 MAPK in cells by sorbitol-induced hyperosmotic stress increased phosphorylation of S-tag-tensin1, which reduced binding to deleted in liver cancer-1 and increased binding to endogenous pTyr proteins, including p130Cas and focal adhesion kinase. These data demonstrate that tensin1 is extensively phosphorylated on Ser/Thr residues in cells and phosphorylation by p38 MAPK regulates the specificity of the tensin1 Src homology 2 domain for binding to different proteins. Tensin1 provides a hub for connecting signaling pathways involving p38 MAP kinase, tyrosine kinases and RhoGTPases.
Highlights
Tensin1 is the archetype of a family of focal adhesion proteins
The C terminus region of tensin1, as well as family members tensin2, tensin3, and c-ten, has adjacent Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains that interact with the tyrosine phosphorylated proteins Dok2 and PDK1 [5] as well as PI3 kinase, p130Cas, and focal adhesion kinase (FAK) [6], thereby posing a role for tensin1 in multiple signal transduction pathways
Mass analyses were completed on LTQ-FT-ion cyclotron resonance and LTQ-Orbitrap hybrid instruments using collisionally activated dissociation (CAD) and front end electron transfer dissociation (FETD)
Summary
Tensin1 is the archetype of a family of focal adhesion proteins. Tensin1 has a phosphotyrosine binding domain that binds the cytoplasmic tail of -integrin, a Src homology 2 domain that binds focal adhesion kinase, p130Cas, and the RhoGAP called deleted in liver cancer-1, a phosphatase and tensin homology domain that binds protein phosphatase-1␣ and other regions that bind F-actin. When cells expressing S-tag tensin1 were treated with calyculin A to inhibit Ser/Thr phosphatases, we identified 12 additional Ser/ Thr phosphosites (Fig. 1B) for a total of 62 sites (see Supplemental Table 2). Treatment of cells with calyculin A produced a noticeable reduction in electrophoretic mobility of S-tag tensin1 in SDS-PAGE (see Fig. 3A), corresponding to phosphorylation of these additional sites.
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