No abstract

Platelets express a single low affinity receptor for immunoglobulin, Fc␥RII, that triggers multiple cellular responses upon interaction with multivalent immune complexes. In this study we show that immobilized IgG is also a potent stimulant of platelet activation triggering adhesion, aggregation, massive dense granule secretion, and thromboxane production. Platelet adhesion to IgG was blocked by the Fc␥RII receptor-specific monoclonal antibody, IV.3. Pretreatment of the platelets with cytochalasin D to inhibit actin polymerization similarly prevented cell binding to IgG having no effect on platelet binding to fibrinogen. Platelet adhesion to IgG also led to the induction of tyrosine phosphorylation of multiple proteins including pp125 FAK and p72 SYK . These proteins were also tyrosine-phosphorylated in ␣ IIb ␤ 3 -deficient IgG-adherent platelets from patients with Glanzmann's thrombasthenia. These data demonstrate that Fc␥RII mediates pp125 FAK phosphorylation and platelet adhesion to IgG independent of the integrin ␣ IIb ␤ 3 . Treatment of the platelets with bisindolylmaleimide to inhibit protein kinase C prevented phosphorylation of pp125 FAK as well as several other proteins, but not p72 SYK phosphorylation. This study establishes that the Fc␥RII receptor mediates pp125 FAK phosphorylation via protein kinase C.Platelets express a single-chain low affinity receptor for immunoglobulin, Fc␥RII (1, 2). Fc␥ receptors expression is restricted to cells of hematopoietic lineage (3). Fc␥ receptor activation has been linked to diverse functions that include activation of tyrosine kinases, elevation of intracellular calcium, and regulation of transcription of genes encoding cytokines (3). In platelets, soluble immune complexes or crosslinking of the Fc␥RII receptor with secondary antibodies trigger a robust activation response that includes changes in intracellular calcium concentration, phosphatidic acid metabolism, and thromboxane production (4, 5). Additional activation responses in suspended platelets, including granule secretion and aggregation, are dependent on thromboxane production (4, 5). Fc␥RII ligation also triggers the induction of tyrosine phosphorylation of multiple cellular proteins (6). These include the Fc␥RII receptor itself (2, 6), a 40-kDa sialoglycoprotein that does not have an intrinsic kinase activity, and the Fc␥RII-associated protein-tyrosine kinase, p72 SYK (2, 6). p72 SYK , a homologue of the T cell-associated protein-tyrosine kinase ZAP-70, is a non-receptor tyrosine kinase containing two SH2 domains but no SH3 domain (7). Clustering of chimeric transmembrane proteins bearing intracellular SYK or its homologue ZAP-70 sequences in T cells is sufficient to trigger calcium mobilization and cytolytic effector functions (8, 9). Similarly, clustering of p72 SYK chimera introduced into rat basophilic leukemia (RBL-2H3) cells is sufficient to trigger cellular responses that include protein tyrosine phosphorylation and synthesis and release of allergic mediators (10). p72 SYK activation may thus prov...

Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNA binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

A mutation that fails to complement certain alleles of the testis-specific [32-tubulin gene (B2t) of Drosophila melanogaster maps to a separate locus, haywire, located at 3-34.4 map units in polytene region 67E3-F3. Second-site non-complementing mutations such as hay ~c2 and B2t alleles could identify genes that encode products that participate in the same functions or that interact in the same structure. Consistent with a structural interaction between the hay gene product and [32-tubulin, the genetic interaction between hay ~2 and B2t requires the presence of the mutant hay gene product; a deficiency for the hay region complements the same alleles of B2t that hay ~2 fails to complement, hay n~2 is a recessive male sterile mutation in a genetic background that is wild type at the B2t locus. Homozygous males have defects in meiosis, flagellar elongation and nuclear shaping, the three major microtubule-based processes in which the testis-specific [32-tubulin participates. The hay ~z allele also has effects outside of spermatogenesis. It is a temperature-sensitive semilethal mutation, and homozygous hay ~2 females have reduced fertility. These phenotypes are consistent with a role for the haywire gene product in general microtubule function. Analysis of second-site noncomplementing mutations such as hay ~2 offers a genetic tool for analysis of interacting proteins in complex assemblies.

This study identified a number of variables that may be useful in clinical screening for vulnerability to physical decline.