IDENTIFICATION OF A NOVEL POLYPROLINE RECOGNITION SITE IN THE CYTOSKELETAL-ASSOCIATED PROTEIN, PROLINE SERINE THREONINE PHOSPHATASE INTERACTING PROTEIN

Protein-protein interactions are often mediated by the recognition of proline-rich domains by SH3 or WW modules, Previously, we demonstrated that the PEST type protein-tyrosine phosphatase, PTP HSCF (hematopoie tic stem cell fraction), bound to a novel cytoskeletal associated prot ein, proline serine threonine phosphatase interacting protein (PST PIP ), via an interaction between the proline-rich COOH terminus of the PT P and a site within the putative coiled-coil domain of PST PIP, Here w e describe a more detailed analysis of this interaction, Earlier data suggested that the NH2, terminus of PST PIP was important for binding to the phosphatase, and deletion of the NH2-terminal 50 amino acids of the PST PIP resulted in an apparently misfolded protein that was inca pable of binding PTP HSCF. To examine the region involved with binding to PTP HSCF, alanine-scanning mutants were produced at intervals thro ughout PST PIP., This analysis demonstrated that a tryptophan at posit ion 232 was essential for binding in vitro., Transfection experiments demonstrated that the Trp(232) mutant protein was capable of associati on with the cortical cytoskeleton but was not bound to PTP HSCF in viv o., Alanine scanning of a peptide derived from the COOH-terminal proli ne-rich domain of PTP HSCF revealed that a subset of prolines, as well as other residues, was required for efficient binding to PST PIP, and introduction of alanines at some of these positions in the protein re sulted in decreased binding to PST PIP in vitro and in vivo, Analysis of in vivo tyrosine phosphorylation of the Trp(232) mutant of PST PIP in the presence of v-Src revealed that this protein was phosphorylated more efficiently than the wild-type molecule, Thus, the interaction b etween PTP HSCF and PST PLP is mediated by a novel site in the cytoske letal associated protein which interacts with residues within the prol ine-rich COOH terminus of the phosphatase.