Malinin N

Malinin N. on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr482 or Ser484 was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476C485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin 3 activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs. (7). In mammals, there are three kindlin family members, each characterized by a FERM domain bisected by a pleckstrin homology domain. The FERM domains of kindlins are most closely related to the FERM domain of talin, which is also involved in regulation of integrin signaling (8,C12). Kindlins and talin bind to the cytoplasmic tails of integrin subunits via their F3 (phosphotyrosine binding) subdomains within their FERM domains. However, the binding sites of talin and kindlins within FIGF the -cytoplasmic tails do not overlap (6, 13), and the two interactions appear to act cooperatively to optimize integrin activation (13, 14). Hence, cells or mice with decreased kindlin expression levels are unable to properly activate their integrins. Kindlin-1 is expressed predominantly in epithelial cells, and mutation in the kindlin-1 gene causes Kindler syndrome in humans (15, 16), a rare disease characterized by skin blistering, poikiloderma with frequent intestinal complications. These phenotypes are recapitulated in mice in which the kindlin-1 gene has been inactivated (17). Kindlin-2 is expressed in most tissues and in many different cell types, and knock-out of kindlin-2 is lethal in mice and zebrafish during embryonic development (14, 18). Mice in which the kindlin-3 gene has been inactivated display defects in platelet (19) and leukocyte (20) responses dependent on integrin activation, and the mice die by day 7 postnatally (19). Kindlin-3 mutations in have been Pristinamycin identified in humans with a rare syndrome referred to as LADIII (21,C25) with manifestations that include episodic bleeding, susceptibility to frequent infections Pristinamycin and osteopetrosis, which are consequences of an inability to activate 1, 2 and 3 integrin (22, 23, 25), and variably in abnormal red cell shapes (25). Kindlin-3 is also present and functional in endothelial cells (26) and breast cancer cells (27), where it acts as a tumor promoter (27). Despite this ample evidence emphasizing the role of kindlin-3 in integrin function in variety of cells, the mechanisms underlying Pristinamycin kindlin-mediated integrin activation are largely unknown. Recently, it has been established that the calpain I cleavage of kindlin-3 at Tyr373 controls the dynamics of integrin/kindlin-3 interaction and, in turn, integrin-dependent adhesion and migration of hematopoietic cells (28). The other Pristinamycin known functional site in kindlin-3 is its integrin-binding site in its F3 domain that centers at Gln597/Trp598. Mice in which these two residues have been mutated to alanines are unable to stop bleeding upon tail resection or form arterial occlusions but are viable Pristinamycin for least 6 months (29). Here, we sought to identify possible post-translational modification(s) that regulate kindlin-3 functions in hematopoietic cells. One of the possible mechanisms regulating the ability of kindlins to activate integrins is phosphorylation. Previous work has shown that phosphorylation on the integrin 3 CT2 regulates kindlin-2 binding (30). In this work, we have considered how this post-translational modification might regulate the function of kindlin-3 and have utilized HEL cells and human platelets as model systems. HEL cells are.