title: The Tyrosine Kinase c-Src Specifically Binds to the Active Integrin αIIbβ3 to Initiate Outside-in Signaling in Platelets.
abstract: It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the β3 integrin subunit via its SH3 domain. Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the β3 tail binding site. Accordingly, we have re-examined c-Src binding to β3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and β3. Following platelet activation, however, c-Src was co-immunoprecipitated with β3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the (15)N-labeled SH3 domain induced by the C-terminal β3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the β1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the β3 peptide to the RT- loop. Under these conditions, the β3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the β3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with β3 are weak and insensitive to β3 tail mutations.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158.
authors.forename: Yibing
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Departments of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Span
authors.affiliation: Departments of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Patrik
authors.initials: P
authors.lastname: Nygren
authors.affiliation: Departments of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: David T
authors.initials: DT
authors.lastname: Moore
authors.affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
authors.forename: Hong
authors.initials: H
authors.lastname: Cheng
authors.affiliation: Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104; Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Roder
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158.
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
authors.affiliation: Departments of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address: bennetts@mail.med.upenn.edu.
authors.forename: Joel S
authors.initials: JS
authors.lastname: Bennett
mesh_terms: D001792:Blood Platelets / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D000081247:CSK Tyrosine-Protein Kinase; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human
keywords: Src; Src homology 3 domain (SH3 domain); integrin; platelet; signal transduction