Abstract
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 (hematopoietic stem cell fraction), bound to a novel cytoskeletal associated protein, proline serine threonine phosphatase interacting protein (PST PIP), via an interaction between the proline-rich COOH terminus of the PTP and a site within the putative coiled-coil domain of PST PIP. Here we 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 incapable of binding PTP HSCF. To examine the region involved with binding to PTP HSCF, alanine-scanning mutants were produced at intervals throughout PST PIP. This analysis demonstrated that a tryptophan at position 232 was essential for binding in vitro. Transfection experiments demonstrated that the Trp232 mutant protein was capable of association with the cortical cytoskeleton but was not bound to PTP HSCF in vivo. Alanine scanning of a peptide derived from the COOH-terminal proline-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 resulted in decreased binding to PST PIP in vitro and in vivo. Analysis of in vivo tyrosine phosphorylation of the Trp232 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 between PTP HSCF and PST PIP is mediated by a novel site in the cytoskeletal associated protein which interacts with residues within the proline-rich COOH terminus of the phosphatase.
Highlights
The specific interactions of a large number of intracellular proteins are involved in a diversity of signaling pathways in eukaryotic cells
Dephosphorylation of proline serine threonine phosphatase interacting protein (PST PIP) phosphotyrosines by protein-tyrosine phosphatases (PTPs) HSCF required the presence of a proline-rich region that is ϳ20 amino acids long in the COOH terminus of the phosphatase, and it was demonstrated that this region appeared to be necessary and sufficient to mediate binding between these two proteins
All of the clones isolated in this procedure began within 10 –15 amino acids of the NH2 terminus of PST PIP, consistent with the proposal that the NH2 terminus was critical for binding to PTP HSCF
Summary
The specific interactions of a large number of intracellular proteins are involved in a diversity of signaling pathways in eukaryotic cells. Dephosphorylation of PST PIP phosphotyrosines by PTP HSCF required the presence of a proline-rich region that is ϳ20 amino acids long in the COOH terminus of the phosphatase, and it was demonstrated that this region appeared to be necessary and sufficient to mediate binding between these two proteins. Examination of this COOH-terminal region revealed that it bore a striking resemblance to the proline-rich motifs recognized by SH3 domains, consistent with its interacting with the PST PIP SH3 module [7,8,9]. These data define a novel type of protein-protein interaction which appears to regulate the levels of tyrosine phosphorylation of a cytoskeletal associated protein
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
