ShcA is an important mediator of Ras/ MAPK activation in PTK-regulated pathways triggered by surface receptors. This function is subserved by the constitutively expressed p52-kDa isoform. Besides activating Ras, p52Shc couples the TCR to Rho GTPases, and thereby participates in actin cytoskeleton remodeling in T cells. Here we have addressed the potential involvement of p52Shc in T-cell chemotaxis and the role of the phosphorylatable tyrosine residues, YY239/240 and Y317, in this process. We show that CXCR4 engagement by the homeostatic chemokine, SDF-1␣, results in p52Shc phosphorylation and its assembly into a complex that includes Lck, ZAP-70, and Vav. This process was found to be both Lck and Gi dependent. Expression of p52Shc mutants lacking YY239/240 or Y317, or p52Shc deficiency, resulted in a profound impairment in CXCR4 signaling and SDF-1␣-dependent chemotaxis, underscoring a crucial role of p52Shc as an early component of the CXCR4 signaling cascade. p52Shc was also found to be required for ligand-dependent CXCR4 internalization independently of tyrosine phosphorylation. Remarkably, CXCR4 engagement promoted phosphorylation of the chain of the TCR/CD3 complex, which was found to be essential for CXCR4 signaling, as well as for SDF-1␣-dependent receptor endocytosis and che IntroductionBy controlling leukocyte trafficking and homing to specific microenvironments, chemokines and their receptors represent a strategic element in the regulation of immune responses. 1 Initially implicated in directing transmigration of neutrophils and other cellular components of the innate immune system to inflamed tissues, chemokines have been found to coordinate temporally and topologically the directional migration and positioning of leukocytes within lymphoid organs and tissues not only in inflammation, but also in homeostatic conditions, thereby participating both in hematopoietic cell differentiation in central lymphoid organs and in the initiation of adaptive immune responses in peripheral lymphoid organs. Although the distinction has become less clear-cut, chemokines have been classified on the basis of their primary function as inflammatory and homeostatic. Among the latter is SDF-1␣/ CXCL12, which interacts with CXCR4. In the hematopoietic system, CXCR4 controls stem-cell migration to the bone marrow and retention of B-cell and granulocytic precursors within this microenvironment, thereby permitting their differentiation. Furthermore, SDF-1␣ is a potent chemoattractant for mature T cells, monocytes, and neutrophils. 2 CXCR4 has also been implicated in brain and heart development and angiogenesis, as well as in some pathological conditions, including tumor metastasis and joint infiltration by inflammatory CD4 ϩ cells in rheumatoid arthritis. 3 Like other chemokine receptors, CXCR4 is a 7-spanning transmembrane receptor coupled to a pertussis toxin (PTX)-sensitive heterotrimeric Gi protein, which, by inhibiting the activity of adenylate cyclase, modulates the levels of intracellular cAMP. 4 This second messenger re...

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Here, we report the identification of three novel missense mutations in the calsequestrin-1 (CASQ1) gene in four patients with tubular aggregate myopathy. These CASQ1 mutations affect conserved amino acids in position 44 (p.(Asp44Asn)), 103 (p.(Gly103Asp)), and 385 (p.(Ile385Thr)). Functional studies, based on turbidity and dynamic light scattering measurements at increasing Ca concentrations, showed a reduced Ca -dependent aggregation for the CASQ1 protein containing p.Asp44Asn and p.Gly103Asp mutations and a slight increase in Ca -dependent aggregation for the p.Ile385Thr. Accordingly, limited trypsin proteolysis assay showed that p.Asp44Asn and p.Gly103Asp were more susceptible to trypsin cleavage in the presence of Ca in comparison with WT and p.Ile385Thr. Analysis of single muscle fibers of a patient carrying the p.Gly103Asp mutation showed a significant reduction in response to caffeine stimulation, compared with normal control fibers. Expression of CASQ1 mutations in eukaryotic cells revealed a reduced ability of all these CASQ1 mutants to store Ca and a reduced inhibitory effect of p.Ile385Thr and p.Asp44Asn on store operated Ca entry. These results widen the spectrum of skeletal muscle diseases associated with CASQ1 and indicate that these mutations affect properties critical for correct Ca handling in skeletal muscle fibers.

The constitutive/inducible association of the T cell receptor (TCR) with isolated detergent-resistant, lipid raft-derived membranes has been studied in Jurkat T lymphocytes. Membranes resistant to 1% Triton X-100 contained virtually no CD3⑀, part of the TCR complex, irrespective of cell stimulation. On the other hand, membranes resistant either to a lower Triton X-100 concentration (i.e. 0.2%) or to the less hydrophobic detergent Brij 58 (1%) contained (i) a low CD3⑀ amount (approximate 2.7% of total) in resting cells and (ii) a several times higher amount of the TCR component, after T cell stimulation with either antigen-presenting cells or with phytohemagglutinin. It appeared that CD3/TCR was constitutively associated with and recruited to a raft-derived membrane subset because (i) all three membrane preparations contained a similar amount of the raft marker tyrosine kinase Lck but no detectable amounts of the conventional membrane markers, CD45 phosphatase and transferrin receptor; (ii) a larger amount of particulate membranes were resistant to solubilization with 0.2% Triton X-100 and Brij 58 than to solubilization with 1% Triton X-100; and (iii) higher cholesterol levels were present in membranes resistant to either the lower Triton X-100 concentration or to Brij 58, as compared with those resistant to 1% Triton X-100. The recruitment of CD3 to the raft-derived membrane subset appeared (i) to occur independently of cell signaling events, such as protein-tyrosine phosphorylation and Ca 2؉ mobilization/influx, and (ii) to be associated with clustering of plasma membrane rafts induced by multiple cross-linking of either TCR or the raft component, ganglioside GM 1 . We suggest that during T cell stimulation a lateral reorganization of rafts into polarized larger domains can determine the recruitment of TCR into these domains, which favors a polarization of the signaling cascade.

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