Regulation of protein tyrosine phosphatase-1B enzymatic activity: The roles of cell adhesion, tyrosine residue 66, the C-terminal region, and the first proline-rich region

The reversible phosphorylation of proteins on specific tyrosine residues is a fundamental aspect of many signal transduction pathways and is controlled by the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). While significant progress has been made toward understanding the regulation of PTKs, there is still much to be learned about the regulation of PTPs. In this study, evidence is presented that the catalytic activity of the nonreceptor type PTP, PTP1B, is modulated by changes in cell adhesion to extracellular matrix proteins. Placing cells in suspension for 20 minutes lead to a significant increase in PTP1B activity compared to that from cells left adherent. This increase was reversible by replating suspended cells onto fibronectin or collagen I for 20 minutes. Additionally, neither p130cas nor Src, Yes, and Fyn, were necessary for the inhibition of PTP1B activity by cell adhesion.; To identify the residues important for this effect, various mutated forms of PTP1B were expressed in cells and then assayed for activity. CTD-PTP1B, truncated to remove residues 322-435, was distinctly more active than WT-PTP1B, suggesting that the C-terminal region might normally limit catalytic activity. However, this region was not required for the adhesion regulation, as the activity of CTD-PTP1B was still inhibited when suspended cells were replated onto collagen I. PA-PTP1B, in which prolines 309 and 310 were substituted with alanines, increased activity when cells were placed in suspension. However, the activity of this mutant was not inhibited by cell adhesion to collagen I for 20 minutes, pointing to a role for the first proline-rich region in PTP1B regulation. Analysis of the Y66F-PTP1B mutant revealed that, rather than being involved in adhesion regulation, residue Y66 might instead be important for the maintenance of PTP1B thermostability. In soluble PTP assays, the activity of Y66F-PTP1B was similar to that of WT-PTP1B at room temperature but greatly reduced at 37°C.; This study is the first report of the regulation of PTP1B activity by changes in cell adhesion. Such findings might have significant implications in terms of understanding the mechanisms of signal 'crosstalk' between cell adhesion and growth factor receptors.