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Vav in natural killer cells is tyrosine phosphorylated upon cross-linking of Fc gamma RIIIA and is constitutively associated with a serine/threonine kinase.

ABSTRACT: Cross-linking of Fc gamma RIIIA (CD16) receptor on natural killer (NK) cells induces receptor-associated tyrosine kinase activation and tyrosine phosphorylation of numerous intracellular proteins, including phospholipase C (PLC)-gamma 1, PLC-gamma 2 and the associated zeta chain. Here we report that Vav, a proto-oncogene, also became tyrosine phosphorylated upon stimulation of CD16 in interleukin 2-activated NK cells (LAK-NK) as well as in an NK cell line, NK3.3. In addition, we observed that in LAK-NK cells, Vav was associated with a 70 kDa protein that also became tyrosine phosphorylated upon CD16 cross-linking. The association of this 70 kDa protein with Vav was disrupted by ionic detergent treatment. Tyrosine phosphorylation of Vav was inhibited by herbimycin A, a specific tyrosine kinase inhibitor. In vitro kinase assays with Vav immunoprecipitates derived from NK3.3 cells or LAK-NK cells resulted in the appearance of a phosphorylated 58 kDa protein, suggesting the presence of a kinase within the Vav immunoprecipitates. Cross-linking of CD16 did not enhance this Vav-associated kinase activity. Phosphoamino acid analysis of the 58 kDa protein revealed that it was phosphorylated only on serine and threonine residues, indicating that an unidentified serine/threonine kinase is constitutively associated with Vav. These observations suggest that the downstream signalling events regulated by Vav and its associated proteins are complex involving both tyrosine kinases as well as the yet unidentified serine/threonine kinase in NK cells.

SUBMITTER: Xu X

PROVIDER: S-EPMC1217652 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Exposure of the developing brain to immune mediators, including antibodies, is postulated to increase risk for neurodevelopmental disorders and neurodegenerative disease. It has been suggested that immunoglobulin G-immune complexes (IgG-IC) activate Fc gamma receptors (Fc?R) expressed on neurons to modify signaling events in these cells. However, testing this hypothesis is hindered by a paucity of data regarding neuronal Fc?R expression and function. METHODS:Fc?R transcript expression in the hippocampus, cortex, and cerebellum of neonatal male and female rats was investigated ex vivo and in mixed cultures of primary hippocampal and cortical neurons and astrocytes using quantitative PCR analyses. Expression at the protein level in mixed cultures of primary hippocampal and cortical neurons and astrocytes was determined by immunocytochemistry, western blotting, proteotype analysis, and flow cytometry. The functionality of these receptors was assessed by measuring changes in intracellular calcium levels, Erk phosphorylation, and IgG internalization following stimulation with IgG-immune complexes. RESULTS:FcgrIa, FcgrIIa, FcgrIIb, FcgrIIIa, and Fcgrt transcripts were detectable in the cortex, hippocampus, and cerebellum at postnatal days 1 and 7. These transcripts were also present in primary hippocampal and cortical cell cultures, where their expression was modulated by IFN?. Expression of Fc?RIa, Fc?RIIb, and Fc?RIIIa, but not Fc?RIIa or FcRn proteins, was confirmed in cultured hippocampal and cortical neurons and astrocytes at the single cell level. A subpopulation of these cells co-expressed the activating Fc?RIa and the inhibitory Fc?RIIb. Functional analyses demonstrated that exposure of hippocampal and cortical cell cultures to IgG-IC increases intracellular calcium and Erk phosphorylation and triggers Fc?R-mediated internalization of IgG. CONCLUSIONS:Our data demonstrate that developing neurons and astrocytes in the hippocampus and the cortex express signaling competent Fc?R. These findings suggest that IgG antibodies may influence normal neurodevelopment or function via direct interactions with Fc?R on non-immune cells in the brain.

Project description:Activation of human platelets by cross-linking of the platelet low-affinity IgG receptor, the Fc gamma receptor IIA (Fc gamma-RIIA), or by collagen is associated with rapid phosphorylation on tyrosine of the non-receptor tyrosine kinase syk. Phosphorylation is still observed, albeit sometimes reduced, in the presence of a combination of a protein kinase C inhibitor, Ro 31-8220, and the intracellular calcium chelator, BAPTA-AM, demonstrating independence from phosphoinositide-specific phospholipase C (PLC) activity. In contrast, the combination of Ro 31-8220 and BAPTA-AM completely inhibits phosphorylation of syk in thrombin-stimulated platelets. Phosphorylation of syk increases its autophosphorylation activity measured in a kinase assay performed on syk immunoprecipitates. Fc gamma-RIIA also undergoes phosphorylation in syk immunoprecipitates from platelets activated by cross-linking of Fc gamma-RIIA but not by collagen, suggesting that it associates with the kinase. Consistent with this, tyrosine-phosphorylated Fc gamma-RIIA is precipitated by a glutathione S-transferase (GST) fusion protein containing the tandem src homology (SH2) domains of syk from Fc gamma-RIIA- but not collagen-activated cells. Two uncharacterized tyrosine-phosphorylated proteins of 40 and 65 kDa are uniquely precipitated by a GST fusion protein containing the tandem syk-SH2 domains in collagen-stimulated platelets. A peptide based on the antigen recognition activation motif (ARAM) of Fc gamma-RIIA, and phosphorylated on the two tyrosine residues found within this region, selectively binds syk from lysates of resting platelets; this interaction is not seen with a non-phosphorylated peptide. Kinase assays on Fc gamma-RIIA immunoprecipitates reveal the constitutive association of an unidentified kinase activity in resting cells which phosphorylates a 67 kDa protein. Syk is not detected in Fc gamma-RIIA immunoprecipitates from resting cells but associates with the receptor following activation and, together with Fc gamma-RIIA, is phosphorylated in the kinase assay in vitro. These results demonstrate that syk is activated by Fc gamma-RIIA cross-linking and collagen, independent of PLC, suggesting that it may have an important role in the early events associated with platelet activation. The association of syk with Fc gamma-RIIA appears to be mediated through the tandem SH2 domains in syk and the ARAM motif of Fc gamma-RIIA. A similar interaction may underlie the response to collagen, suggesting that its signalling receptor contains an ARAM motif.

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Vav in natural killer cells is tyrosine phosphorylated upon cross-linking of Fc gamma RIIIA and is constitutively associated with a serine/threonine kinase.

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