A Collagen-Like Peptide Stimulates Tyrosine Phosphorylation of syk and Phospholipase Cγ2 in Platelets Independent of the Integrin α2β1

Asselin, J; Gibbins, Jonathan M.; Achison, Marcus; Lee, Young Han; Morton, L F; Farndale, Richard W.; Barnes, Michael J.; Watson, Steve P.

doi:10.1182/blood.v89.4.1235

Cited by 193 publications

(93 citation statements)

References 26 publications

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“…This is supported by induction of PLC-γ tyrosine phosphorylation upon cell adhesion and inhibition of cell spreading in wild-type osteoclasts by a PLC inhibitor. These observations are consistent with previous studies showing that integrin–ECM interactions induce tyrosine phosphorylation of PLC-γ1 (Langholz et al 1997) and PLC-γ2 (Asselin et al 1997). It was also recently reported that phosphorylation of PLC-γ1 at the tyrosine residue 783 is important for regulation of cytoskeletal organization in fibroblasts (Yu et al 1998; Pei and Williamson 1998), while PLC-γ1 can serve as a substrate of c-Src in in vitro kinase assays (Liao et al 1993; Nakanishi et al 1993).…”

Section: Discussionsupporting

confidence: 93%

Convergence of αvβ3Integrin–And Macrophage Colony Stimulating Factor–Mediated Signals on Phospholipase Cγ in Prefusion Osteoclasts

Nakamura

Lipfert

Rodan

et al. 2001

The Journal of Cell Biology

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The macrophage colony stimulating factor (M-CSF) and αvβ3 integrins play critical roles in osteoclast function. This study examines M-CSF– and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130Cas, paxillin, and PLC-γ. However, in response to M-CSF, Src−/− pOCs spread and migrate on Vn in an αvβ3-dependent manner. Involvement of PLC-γ activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF–mediated cell spreading. Furthermore, in Src−/− pOCs M-CSF, together with filamentous actin, causes recruitment of β3 integrin and PLC-γ to adhesion contacts and induces stable association of β3 integrin with PLC-γ, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-γ can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-γ is a common downstream mediator for adhesion and growth factor signals. M-CSF–initiated signaling modulates the αvβ3 integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-γ.

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Section: Discussionsupporting

confidence: 93%

Convergence of αvβ3Integrin–And Macrophage Colony Stimulating Factor–Mediated Signals on Phospholipase Cγ in Prefusion Osteoclasts

Nakamura

Lipfert

Rodan

et al. 2001

The Journal of Cell Biology

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“…P1E6 alone did not have any effect on platelet tyrosine phosphorylation. A similar result has been reported for the effect of the anti-a2 mAb 13, the anti-b1 mAb 6F1 [14] and an undefined anti-a2b1 antibody [36] against collagen-stimulated tyrosine phosphorylation. The magnitude of the shift in the collagen concentration response curves and delay in phosphorylation in the presence of P1E6 and jararhagin is of the same order as for aggregation, suggesting a causal relationship.…”

Section: Jararhagin and P1e6 Inhibit Protein Tyrosine Phosphorylationsupporting

confidence: 86%

“…The prototype of this series of peptides, known as collagen-related peptide (CRP), is based on a glycine-prolinehydroxyproline repeat motif which is crosslinked through cysteine residues at the N-and C-terminals. CRP is a powerful platelet agonist that mimics many of the signalling actions of collagen yet is unable to bind to a2b1 [13,14]. This provides support to the two-site, two step model of platelet activation by collagen that was proposed several years ago [15,16].…”

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confidence: 73%

Evidence against a direct role of the integrin α2β1 in collagen‐induced tyrosine phosphorylation in human platelets

Hers

Berlanga

Tiekstra

et al. 2000

European Journal of Biochemistry

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In the present study we have investigated whether the collagen receptor a2b1 (GPIa-IIa; GP, glycoprotein) regulates protein tyrosine phosphorylation in platelets directly through activation of tyrosine kinases or indirectly through modification of the response to GPVI. The interaction of collagen with a2b1 was inhibited in two distinct ways, using the metalloprotease jararhagin, which cleaves the b1 subunit, or the antibody P1E6 which competes with binding of collagen to the integrin. The two inhibitors caused a shift to the right in the collagen concentration response curves for protein tyrosine phosphorylation and platelet activation consistent with a causal relationship between the two events. There was no change in the overall pattern of tyrosine phosphorylation in response to high concentrations of collagen in the presence of a2b1 blockade demonstrating that the integrin is not required for this event. In contrast, jararhagin and P1E6 had a small, almost negligible inhibitory effect against responses to the GPVI-selective agonist collagen-related peptide (CRP) and the G protein-coupled receptor agonist thrombin. Crosslinking of a2b1 in solution or by adhesion to a monolayer using a variety of antibodies to either subunit of the integrin did not induce detectable protein tyrosine phosphorylation in whole cell lysates. The snake venom toxin trimucytin-stimulated a similar pattern of tyrosine phosphorylation to that induced by crosslinking of GPVI which was maintained in the presence of jararhagin. Trimucytin may therefore induce activation via GPVI rather than a2b1 as previously thought. These observations show that the integrin a2b1 is not required for regulation of tyrosine phosphorylation by collagen.Keywords: collagen; GPIa-IIa; GPVI; platelets; tyrosine phosphorylation.When the subendothelium lining is damaged, platelets adhere to newly exposed collagen fibres and undergo activation leading to thromboxane A 2 formation, aminophospholipid exposure, granule secretion and platelet aggregation. Several surface glycoproteins have been implicated as collagen receptors, including glycoprotein (GP) IV (also known as CD36), GPVI, a recently cloned protein of 65 kDa protein, a 85/90 kDa protein and the integrin a2b1. GPVI and a2b1 are believed to play pivotal roles in the interaction of platelets with collagen as patients deficient in the expression of either protein have impaired collagen±platelet interactions and mild bleeding disorders. The consensus is that the integrin a2b1 plays a critical role in adhesion, whilst GPVI is essential for activation. There is evidence for minor roles of other receptors in these responses (reviewed in [1±3]).GPVI is associated with the Fc receptor g-chain (FcR g-chain) on the platelet surface [4,5], and crosslinking leads to phosphorylation of the FcR g-chain on a sequence known as an immunoreceptor tyrosine-based activation motif (ITAM) [6], probably by a Src kinase [7,8]. This enables binding and activation of the tyrosine kinase Syk, starting a chain of events that leads to t...

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“…Resulting protein complexes and immunoprecipitates were resolved by SDS/PAGE and transferred to poly(vinylidene difluoride) (PVDF) membranes. Immunoblotting was carried out as previously described [25]. Protein detection was by enhanced chemiluminescence.…”

Section: Gst Precipitation Immunoprecipitation and Immunoblottingmentioning

confidence: 99%

Evidence that phospholipase C‐γ2 interacts with SLP‐76, Syk, Lyn, LAT and the Fc receptor γ‐chain after stimulation of the collagen receptor glycoprotein VI in human platelets

Gross

Melford

Watson

1999

European Journal of Biochemistry

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Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor g-chain (FcR g-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C-g 2 (PLC-g 2). In this study tyrosine-phosphorylated proteins that associate with PLC-g 2 after stimulation by a collagen-related peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-g2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC-g 2. The majority of the tyrosinephosphorylated proteins that associate with PLC-g 2 bind to its C-terminal SH2 domain. These were found to include PLC-g 2, Syk, SH2-domain-containing leucocyte protein of 76 kDa (SLP-76), Lyn, linker for activation of T cells (LAT) and the FcR g-chain. Direct association was detected between PLC-g 2 and SLP-76, and between PLC-g2 and LAT upon CRP stimulation of platelets by far-Western blotting. FcR g-chain and Lyn were found to co-immunoprecipitate with PLC-g2 as well as with unidentified 110-kDa and 75-kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-g2 in platelets is in contrast with that for PLC-g1 and Syk in B cells. The in vivo function of PLC-g2 SH2 domains was examined through measurement of Ca 2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C-terminal SH2 domain is involved in the regulation of PLC-g2. These data indicate that the C-terminal SH2 domain of PLC-g2 is important for PLC-g2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR g-chain.

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A Collagen-Like Peptide Stimulates Tyrosine Phosphorylation of syk and Phospholipase Cγ2 in Platelets Independent of the Integrin α2β1

Cited by 193 publications

References 26 publications

Convergence of αvβ3Integrin–And Macrophage Colony Stimulating Factor–Mediated Signals on Phospholipase Cγ in Prefusion Osteoclasts

Convergence of αvβ3Integrin–And Macrophage Colony Stimulating Factor–Mediated Signals on Phospholipase Cγ in Prefusion Osteoclasts

Evidence against a direct role of the integrin α2β1 in collagen‐induced tyrosine phosphorylation in human platelets

Evidence that phospholipase C‐γ2 interacts with SLP‐76, Syk, Lyn, LAT and the Fc receptor γ‐chain after stimulation of the collagen receptor glycoprotein VI in human platelets

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