Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage–specific FAK knockout

Abstract

IntroductionFocal adhesion kinase (FAK) is an essential nonreceptor protein tyrosine kinase that is expressed ubiquitously and is conserved in mammals and lower eukaryotic organisms. [1][2][3][4] The principal FAK stimulus is integrin engagement (Guan and Shalloway 5 and reviewed in Parsons 6 ), although its direct interaction with, and activation via, platelet-derived and epidermal growth factor receptors suggests that it may also function downstream of other membrane-bound growth factor and cytokine receptors. 7 Autophosphorylation of FAK Tyr397 occurs rapidly following integrin activation, and the resulting phosphorylated residue acts as a docking site for the SH2 domains of Src-family kinases. 8,9 FAK catalytic activity is increased by subsequent Src-mediated phosphorylation of FAK residues Tyr576 and Tyr577, 10 followed by Tyr861 and Tyr925, which act as binding sites for the SH3 domain of p130CAS and the SH2 domain of the adaptor protein GRB2, respectively. 11,12 Tyr925, located in the focal adhesion targeting domain, also mediates interactions with integrin-associated proteins such as talin and paxillin, 13 thereby recruiting FAK to focal adhesion sites.Activated FAK modulates the activity of a broad range of downstream signaling proteins, including phosphoinositide 3-kinase (PI3-K) 14 and phospholipase C (PLC)-␥, 15 as well as a number of small GTPases such as Ras, Rac, and Rho (reviewed in 16 ). Extensive studies indicate that FAK is essential for normal cell migration. FAK-deficient cells migrate poorly in response to chemokines; form an increased number of prominent "immature" focal adhesions, apparently due to decreased focal adhesion turnover; and do not spread normally on extracellular matrices (ECMs). 17,18 Megakaryopoiesis and platelet production are tightly regulated by a number of growth factors and cytokines to maintain a normal number of circulating platelets. The principal regulator of megakaryopoiesis is thrombopoietin (Tpo), 19 although other fac 1 tors such as interleukin-3 and stem-cell factor work in synergy with Tpo during the earlier stages of megakaryocytic progenitor cell expansion. 20 It has become apparent that the microenvironments in which megakaryocytes function are also of critical importance to megakaryopoiesis. 21,22 Indeed, direct cell-cell and cell-ECM interactions have been demonstrated to influence megakaryocyte differentiation and proplatelet formation. [23][24][25] Although FAK is important in regulating cell spreading and migration in response to integrin-ECM interactions, the role of FAK in megakaryopoiesis remains unclear, partly because Fak deletion in mice is lethal at embryonic day 8.5, before the onset of significant definitive hematopoiesis. Although the role of proline-rich tyrosine kinase-2 (PYK2), which shares sequence homology and similar characteristics with FAK, has been characterized in megakaryocytes, 8,[26][27][28] its localization and dependence on intracellular calcium make the 2 proteins functionally different. 29 FAK activation in platelets require...

PKCθ is dispensable for megakaryopoiesis

“…injection of 50 mg/kg anti-mouse CD41 antibody, and monitored recovery. After twenty-four hours we observed greater than 85% platelet reduction in both PKCy À/À and WT littermate control mice, which is consistent with previous reports [16,17]. However, recovery up to 7 days was unaltered with PKCy deficiency suggesting that PKCy does not contribute to in vivo megakaryopoiesis or platelet production ( Figure 2).…”

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

“…Whether these platelets contain full-length FlnA or a truncated form, as shown for talin (Priddle et al, 1998), remains to be investigated. The high efficiency of gene inactivation is similar to that described for the widely used megakaryocytespecific Pf4-Cre transgenic mouse and posits the GATA1-Cre mouse as a strong alternative for studies in megakaryocytes and platelets (Léon et al, 2007;Petrich et al, 2007;Tiedt et al, 2007;Hitchcock et al, 2008;Wen et al, 2009).…”

FAK regulates platelet extravasation and tumor growth after antiangiogenic therapy withdrawal

“…Platelet expression of focal adhesion kinase is crucial for platelet migration. An important regulator of shape change and movement of platelets is focal adhesion kinase (FAK, also known as protein tyrosine kinase 2 [PTK2]) (33). FAK is overexpressed in a variety of human cancers (34) and is at the interface of tumor cells and their microenvironment (35).…”

“…Another possible explanation as to why tumor weights were not significantly different is that the time point of necropsy was dependent on the group of WT mice in which antiangiogenic therapy was withdrawn; these mice became moribund much earlier than the Pf4-Cre + Fak fl/fl mice. The Pf4-Cre + Fak fl/fl system was published and characterized earlier (33). The authors evaluated the expression of FAK in megakaryocytes and platelets as well as in several other organs.…”