Activation of Syk protein tyrosine kinase through interaction with integrin β cytoplasmic domains

AbstractSyk protein tyrosine kinase is essential for immune system development and function [1] and for the maintenance of vascular integrity [2, 3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs) [1]. Syk can also be activated by integrin adhesion receptors [4, 5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin β3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the β3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the β3 cytoplasmic tail [β3(759X)] decreased Syk binding and disrupted its physical association with integrin αIIbβ3. Furthermore, cells expressing αIIbβ3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the αIIbβ3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin β3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase

Coordinate interactions of Csk, Src, and Syk kinases with αIIbβ3 initiate integrin signaling to the cytoskeleton

Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin αIIbβ3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in αIIbβ3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with αIIbβ3. However, fibrinogen binding caused Csk to dissociate from αIIbβ3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with αIIbβ3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to αIIbβ3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton

Differential Requirement for LAT and SLP-76 in GPVI versus T Cell Receptor Signaling

Mice deficient in the adaptor Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) exhibit a bleeding disorder and lack T cells. Linker for activation of T cells (LAT)-deficient mice exhibit a similar T cell phenotype, but show no signs of hemorrhage. Both SLP-76 and LAT are important for optimal platelet activation downstream of the collagen receptor, GPVI. In addition, SLP-76 is involved in signaling mediated by integrin αIIbβ3. Because SLP-76 and LAT function coordinately in T cell signal transduction, yet their roles appear to differ in hemostasis, we investigated in detail the functional consequences of SLP-76 and LAT deficiencies in platelets. Previously we have shown that LAT−/− platelets exhibit defective responses to the GPVI-specific agonist, collagen-related peptide (CRP). Consistent with this, we find that surface expression of P-selectin in response to high concentrations of GPVI ligands is reduced in both LAT- and SLP-76–deficient platelets. However, platelets from LAT−/− mice, but not SLP-76−/− mice, aggregate normally in response to high concentrations of collagen and convulxin. Additionally, unlike SLP-76, LAT is not tyrosine phosphorylated after fibrinogen binding to integrin αIIbβ3, and collagen-stimulated platelets deficient in LAT spread normally on fibrinogen-coated surfaces. Together, these findings indicate that while LAT and SLP-76 are equally required for signaling via the T cell antigen receptor (TCR) and pre-TCR, platelet activation downstream of GPVI and αIIbβ3 shows a much greater dependency on SLP-76 than LAT

Post-discharge prognosis of patients admitted to hospital for heart failure by world region, and national level of income and income disparity (REPORT-HF): a cohort study

Background: Heart failure is a global public health problem, affecting a large number of individuals from low-income and middle-income countries. REPORT-HF is, to our knowledge, the first prospective global registry collecting information on patient characteristics, management, and prognosis of acute heart failure using a single protocol. The aim of this study was to investigate differences in 1-year post-discharge mortality according to region, country income, and income inequality. Methods: Patients were enrolled during hospitalisation for acute heart failure from 358 centres in 44 countries on six continents. We stratified countries according to a modified WHO regional classification (Latin America, North America, western Europe, eastern Europe, eastern Mediterranean and Africa, southeast Asia, and western Pacific), country income (low, middle, high) and income inequality (according to tertiles of Gini index). Risk factors were identified on the basis of expert opinion and knowledge of the literature. Findings: Of 18 102 patients discharged, 3461 (20%) died within 1 year. Important predictors of 1-year mortality were old age, anaemia, chronic kidney disease, presence of valvular heart disease, left ventricular ejection fraction phenotype (heart failure with reduced ejection fraction [HFrEF] vs preserved ejection fraction [HFpEF]), and being on guideline-directed medical treatment (GDMT) at discharge (p<0·0001 for all). Patients from eastern Europe had the lowest 1-year mortality (16%) and patients from eastern Mediterranean and Africa (22%) and Latin America (22%) the highest. Patients from lower-income countries (ie, ≤US$3955 per capita; hazard ratio 1·58, 95$12 235 per capita) or lower income inequality (ie, from the lowest Gini tertile). Compared with patients with HFrEF, patients with HFpEF had a lower 1-year mortality with little variation by income level (pinteraction for HFrEF vs HFpEF <0·0001). Interpretation: Acute heart failure is associated with a high post-discharge mortality, particularly in patients with HFrEF from low-income regions with high income inequality. Regional differences exist in the proportion of eligible patients discharged on GDMT, which was strongly associated with mortality and might reflect lack of access to post-discharge care and prescribing of GDMT. Funding: Novartis Pharma

Severe aortic valve stenosis and nosebleed

Aortic valve stenosis is known to be associated with loss of high molecular von Willebrand multimers. This can lead to gastrointestinal bleeding in patients with gastrointestinal angiodysplasia, the Heyde syndrome. Here we present a case of anaemia and severe epistaxis associated with acquired von Willebrand syndrome. Gastrointestinal endoscopy revealed no bleeding source. Calcifying aortic stenosis was confirmed by echocardiography. Loss of high molecular weight multimers of von Willebrand factor in our patient was shown by immunoblot analysis. If severe epistaxis occurs in the context of symptomatic aortic valve stenosis, it might be an additional reason to recommend valve replacement surgery to the patient. (c) 2006 Elsevier Ireland Ltd. All rights reserved

Regulation of outside-in signaling in platelets by integrin-associated protein kinase C beta

10 páginas, 9 figuras -- PAGS nros. 644-653Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin αIIbβ3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. αIIbβ3 interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether αIIbβ3 might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKCβ co-immunoprecipitated with αIIbβ3 in response to the interaction of platelets with soluble or immobilized fibrinogen. PKCβ recruitment to αIIbβ3 was accompanied by a 9-fold increase in PKC activity in αIIbβ3 immunoprecipitates. RACK1, an intracellular adapter for activated PKCβ, also co-immunoprecipitated with αIIbβ3, but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKCβ recruitment to αIIbβ3 and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKCβ spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKCβI to associate with αIIbβ3 and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the β3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to β3. These studies demonstrate that the interaction of αIIbβ3 with activated PKCβ is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through αIIbβ3. Furthermore, the studies extend the concept of αIIbβ3 as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.In addition to their roles in cell adhesion, integrins transmit signals in both directions across the plasma membrane to regulate cytoskeletal organization, motility, and other anchorage-dependent cellular responses (1). In platelets, for example, αIIbβ3 responds to “inside-out” signals with an increase in affinity for cognate ligands such as fibrinogen that bridge platelets to each other and mediate platelet adhesion to sites of vascular damage. In turn, ligand binding to αIIbβ3 triggers outside-in signals that promote cytoskeletal changes necessary for full platelet aggregation and spreading (2, 3). Bidirectional αIIbβ3 signaling is controlled, in part, by specific intracellular proteins that interact with the relatively short cytoplasmic tails of αIIb or β3. For example, binding of talin to β3 is a final common step in the cellular modulation of αIIbβ3 affinity (4), and binding of c-Src and Syk protein-tyrosine kinases to β3 is required for platelet spreading on fibrinogen (5, 6). Several other intracellular proteins, for example, CIB and β3-endonexin, can also bind to αIIb or β3 tails, respectively, and may influence αIIbβ3 functions (7-9). However, the full complement of intracellular proteins that are capable of interacting directly or indirectly with αIIbβ3 is unknown. The protein kinase C (PKC)1 subfamily of AGC serine/threonine kinases has been implicated in integrin function or dynamics in many cell types (10). In platelets, PKC is thought to regulate αIIbβ3 affinity, based on the stimulatory effects of phorbol esters, which bind to PKC C1 domains, and the blocking effects of broad spectrum PKC inhibitors (3). However, the lack of specificity of these compounds limits data interpretation and does not permit conclusions about the roles of specific PKC isoforms (11). PKCs have been categorized as classical (diacylglycerol- and Ca2+-regulated through C1 and C2 domains, respectively), novel (Ca2+-independent but diacylglycerol-regulated), or atypical (Ca2+- and diacylglycerol-independent) (12, 13). Platelets are reported to contain members of all three classes of PKC isozymes (14-21), as well as the related protein kinase D (22). Recently, experimental tools have become available to study specific PKC isoforms in cells, including overexpression, gene targeting and gene knock-down strategies, and molecular imaging (23-25). Some of these tools are potentially relevant to platelets.PKC function depends on the maturation of catalytic activity of the enzyme through phosphorylation and PKC binding to membranes or specific proteins. The latter interactions place PKC in proximity to substrates and relieve autoinhibitory restraints imposed by the binding of a pseudosubstrate sequence to the active site (12, 13, 26). One group of PKC targeting proteins has been termed RACK, which binds selectively to activated PKCs (27). The best characterized protein of this group is RACK1, a 36-kDa protein composed of seven WD40 repeats. RACK1 was originally identified based on its interaction with activated PKCβ and subsequently shown to interact with certain other PKC isoforms and with several other proteins, most notably integrin β cytoplasmic tails and c-Src (see Fig. 1A) (28-30).Given the potential for the cytoplasmic tails of αIIbβ3 to serve as binding sites for signaling molecules and the apparent functional relationships between PKC and αIIbβ3, the present studies were carried out to determine whether specific PKCs associate with αIIbβ3 and if so to determine what the functional relevance of the association is. By using human and mouse platelets and a CHO cell model system, the results show that one particular PKC isoform, PKCβ, inducibly associates with αIIbβ3 in response to fibrinogen binding to cells. The PKCβ/αIIbβ3 interaction appears to be mediated by RACK1 and is required for cytoskeletal reorganization and platelet spreading on fibrinogen, but it is dispensable for the affinity modulation of αIIbβ3This work was supported by National Institutes of Health Grants HL56595, HL57900, and AI38348. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Peer reviewe