Abstract. We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125 F~ (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125 F~ and that pp125 F~ molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays, pp125 F~ was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIbIIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP lib-Ilia. Fibrinogen binding to GP Ub-HIa was not sufficient to induce pp125 F~x phosphorylation because pp125 F~ was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125 s~ is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP lib-Ilia. The induction of tyrosine phosphorylation of pp125 F~ was inhibited in thrombin-and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125 r~ in vivo and stimulation of the phosphorylation of pp125 FA~ in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125 F~ is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125 FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.T HE interactions of cells with extracellular adhesion molecules play critical roles in regulating the morphology, proliferation, migration, and differentiation of cells. One family of receptors for extracellular adhesion molecules, called integrins, is comprised of heterodimeric r transmembrane proteins (1,17,19,20). Although the extracellular interactions between adhesion molecules and their integrin receptors have been well characterized, the interactions of integrins with cytoplasmic targets and their role in signaling pathways that are responsible for adhesion-induced changes in cell behavior are poorly understood (5).Platelets provide a very useful model system for investigating the mechanisms involved in integrin-induced events. The best characterized platelet integrin receptor, GP Hb-HIa, is required for two essential functions of platelets in hemostasis-platelet-to-platelet aggregation and the spreading of J. S. Brugge's present address is Ariad Pharmaceuticals, Inc.,

Proline-rich tyrosine kinase 2 (PYK2) is the main adhesion-induced kinase in bone-resorbing osteoclasts. Previous studies have shown that ligation of ␣ v ␤ 3 integrin in osteoclasts induces c-Src-dependent tyrosine phosphorylation and PYK2 activation, leading to cytoskeletal rearrangement, migration, and polarization of these cells. In this study, we examined the role of PYK2 kinase activity and its major autophosphorylation site in adhesion-dependent signaling and cytoskeletal organization during osteoclast spreading and migration. By infecting pre-fusion osteoclasts using recombinant adenovirus expressing PYK2 and its mutants, we demonstrated that mutation at the autophosphorylation site (Y402F) abolishes PYK2 association with c-Src and reduces significantly phosphorylation at tyrosines 579/ 580 and 881 resulting in inhibition of osteoclast spreading and bone resorption. Overexpression of the kinasedead PYK2(K475A) mutant had no effect on cell spreading, interaction with c-Src, or the phosphorylation level of Tyr-402, Tyr-579/580, and Tyr-881 relative to PYK2(wt)-expressing cells. Taken together these findings suggest that Tyr-402 is the major docking site for c-Src and can be phosphorylated by another tyrosine kinase in osteoclasts but not in HEK293 cells. Interestingly, both PYK2(Y402F) and PYK2(K457A) translocate normally to podosomes and have no effect on macrophage colony-stimulating factor-induced osteoclast migration. Whereas PYK2(Y402F) dominant negatively blocks osteoclast spreading and bone resorption, PYK2(K457A) may function in part as an adaptor by initially recruiting c-Src to the adhesion complex, which appears to activate PYK2 by phosphorylating additional tyrosines in its regulatory and C-terminal domains. We thus concluded that phosphorylation at Tyr-402 in PYK2 is essential in the regulation of adhesion-dependent cytoskeletal organization in osteoclasts. PYK21 is highly expressed in osteoclasts, terminally differentiated bone-resorbing cells of hematopoietic origin, and participates in the signaling initiated by osteoclast interaction with bone. Adhesion to bone matrix induces osteoclast differentiation, cytoskeletal reorganization, and cellular polarization, leading to formation of unique membrane areas for active bone resorption. These include the following: (i) the sealing zone, for tight adhesion to bone matrix; (ii) the ruffled border, for directional secretion of protons and proteases into the resorption lacuna; and (iii) functional secretory domain toward bone marrow space, for transcytosis and release of degraded bone matrix (1-3). ␣ v ␤ 3 integrin is highly expressed in osteoclasts (4) and was suggested to mediate osteoclast adhesion to various bone matrix proteins and to regulate cytoskeletal organization required for migration and formation of the sealing zone (5, 6). Interference with ␣ v ␤ 3 integrin function by blocking antibodies or by RGD-containing peptides or proteins leads to inhibition of osteoclast migration and of bone resorption in vitro and in vivo (6 -12). In additio...

Abstract. Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125 FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin C~m~3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-~3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in otm~3 Proteins of 50-68 kD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen-dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125 FA~. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) t~3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or T4°°-4u). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to O/I~3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125 FAr. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.

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.