Search PubMed⌕ Search

Biomedical subjects

A Marie-Cardine

Publications and source records attributed to A Marie-Cardine.

28 records · Page 2Linked to original sources
PubMed1997

Molecular cloning of SKAP55, a novel protein that associates with the protein tyrosine kinase p59fyn in human T-lymphocytes.

In human T-lymphocytes the Src family protein tyrosine kinase p59(fyn) associates with three phosphoproteins of 43, 55, and 85 kDa (pp43, pp55, and pp85). Employing a GST-Fyn-Src homology 2 (SH2) domain fusion protein pp55 was purified from lysates of Jurkat T-cells. Molecular cloning of the pp55 cDNA reveals that the pp55 gene codes for a so far nondescribed polypeptide of 359 amino acids that comprises a pleckstrin homology domain, a C-terminal SH3 domain, as well as several potential tyrosine phosphorylation sites, among which one fulfills the criteria to bind Src-like SH2 domains with high affinity. Consistent with this observation, pp55 selectively binds to isolated SH2 domains of Lck, Lyn, Src, and Fyn but not to the SH2 domains of ZAP70, Syk, Shc, SLP-76, Grb2, phosphatidylinositol 3-kinase, and c-abl in vitro. Based on these properties the protein was termed SKAP55 (src kinase-associated phosphoprotein of 55 kDa). Northern blot analysis shows that SKAP55 mRNA is preferentially expressed in lymphatic tissues. SKAP55 is detected in resting human T-lymphocytes as a constitutively tyrosine phosphorylated protein that selectively interacts with p59(fyn). These data suggest that SKAP55 represents a novel adaptor protein likely involved in Fyn-mediated signaling in human T-lymphocytes.

Amino Acid Sequence↗
PubMed1996

Recruitment of activated p56lck on endosomes of CD2-triggered T cells, colocalization with ZAP-70.

We have previously established that upon CD2 activation of T cells, p56(lck) showed a transient increase in its kinase activity and was partially internalized. Here we studied the possibility that p56(lck) could retain its kinase activity in the endosomes of CD2-triggered cells. T cells were fractionated on a sucrose gradient, and the endosomal fraction was isolated. In CD2-triggered cells, part of Lck was internalized and presented a maximal kinase activity in the endosome-enriched fraction after 5 min, decreasing thereafter. In the endosomal fraction of activated cells, four tyrosine-phosphorylated proteins of apparent molecular masses of 30, 40, 56, and 70 kDa were detected. We demonstrated that the protein tyrosine kinase ZAP-70 was recruited to the endosomal fraction upon CD2 stimulation with kinetics similar to that of p56(lck), suggesting that recruitment of protein tyrosine kinases to endosomal vesicles could promote specific transduction signals at the intracellular level.

CD2 Antigens↗
PubMed1995

Human T lymphocyte activation induces tyrosine phosphorylation of alpha-tubulin and its association with the SH2 domain of the p59fyn protein tyrosine kinase.

A glutathione-S-transferase-src-homology domain 2 (GST-SH2) fusion protein was employed to identify molecules interacting with the protein tyrosine kinase p59fyn. Among several proteins which bound to the fyn SH2 domain in lysates of human Jurkat T lymphocytes, alpha- and beta-tubulin were identified by N-terminal sequencing. Further analysis established that alpha-tubulin exists as a tyrosine-phosphorylated protein in Jurkat cells, where it interacts with p59fyn, but not with p56lck. By contrast, in untransformed resting human T lymphocytes alpha-tubulin is not detectable as a tyrosine phosphorylated protein. However, following T cell activation, it becomes rapidly phosphorylated on tyrosine residues and subsequently associates with the SH2 domain of fyn. Interestingly, constitutively tyrosine-phosphorylated alpha-tubulin that is able to interact with the fyn-SH2 domain is expressed in peripheral blood T lymphoblasts isolated from leukemic patients in the absence of external stimulation.

Amino Acid Sequence↗
PubMed1995

In vitro binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase.

Although the human immunodeficiency virus type 1 (HIV-1) nef gene still has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1LAI nef gene product was expressed in the bacterial vector pGEX-2T as a glutathione S-transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites in vitro: one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef in vitro. The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis.

Base Sequence↗
PubMed1994

Activation and internalization of p56lck upon CD45 triggering of Jurkat cells.

Relationships between CD45 and p56lck have been suggested by co-immunoprecipitation of both proteins and by dephosphorylation of the p56lck regulatory site, Tyr 505, by CD45 in vitro. We investigated whether the kinase activity of p56lck is modulated in T cells triggered via CD45. We showed that incubation of Jurkat cells with a combination of two anti-CD45 monoclonal antibodies (mAb) (MC5/2 + D3/9) induced an increase in p56lck kinase activity, while a single mAb did not. Under these conditions, p56lck underwent two consecutive waves of activation. This was accompanied by internalization of the kinase and by a time-dependent increased accessibility of CD45 phosphatase at the plasma membrane. Similarly, activation and internalization of p56lck were observed using a combination of anti-CD45 (MC5/2) and anti-CD2 (T11(2)) mAb, suggesting that a functional complex consisting of CD45, CD2 and p56lck was formed upon cell triggering. Taken together, these results suggests that: (i) CD45 participates in the regulation of p56lck kinase activity in vivo and that (ii) CD45 could play a mediator role in the stimulation and endocytosis of p56lck through the CD2 pathway.

Antibodies, Monoclonal↗
PubMed1994

Induction of hyperphosphorylation and activation of the p56lck protein tyrosine kinase by phenylarsine oxide, a phosphotyrosine phosphatase inhibitor.

The T cell protein tyrosine kinase p56lck is implicated in thymic development and mitogenic activation of T lymphocytes, and is itself regulated by reversible tyrosine phosphorylation. When phenylarsine oxide (PAO), a membrane-permeable inhibitor of phosphotyrosine phosphatases, was added to Jurkat T leukemia or LSTRA thymoma cells, the phosphate content of p56lck increased rapidly. The sites of increased phosphorylation were mapped to Tyr-192, Tyr-394 and Tyr-505. Hyperphosphorylated p56lck displayed retarded mobility on SDS gels, unaltered or marginally increased cytoskeletal association, and its catalytic activity changed in a biphasic manner; during the first 10-20 min of PAO-treatment the activity increased and then it declined to very low values within 1-2 hr. Our data suggest that p56lck contains both positive and negative regulatory sites which are constantly dephosphorylated at an unexpectedly high rate by cellular phosphotyrosine phosphatases.

Animals↗
PubMed1994

Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

Amino Acid Sequence↗
PubMed1994

P56lck A lymphocyte specific protein tyrosine kinase: activation, regulation and signal transduction.

p56lck, a src family protein tyrosine kinase interacts with several T cell receptors, like: CD4, CD8, CD2 and the beta-chain of the IL2, thereby receptors devoid of kinase activity may transduce signals via tyr phosphorylation. Tyr 192 and ser 194, located in the SH2 domain of p56lck is phosphorylated upon CD3 triggering, which can change interactions of tyr-P proteins with this SH2 domain. Upon activation through the CD2 or the CD45 receptors the kinase activity of p56lck is temporarily increased. By immunofluorescent and confocal microscopy we observed that a significant proportion of p56lck and CD2 receptors are localized in endosomal vesicles after stimulation. By Western blot we showed a parallel recruitment of the PTK p70-ZAP in this vesicles. The role of p56lck away from the plasma membrane localized in vesicles is under study.

CD2 Antigens↗
PubMed1992

The lymphocyte-specific tyrosine protein kinase p56lck is endocytosed in Jurkat cells stimulated via CD2.

Incubation of the human T cells, Jurkat, with two sets of activating anti-CD2 mAb (T11(2) + T11(3), D66 + T11(1)) induced delocalization of p56lck and CD2 receptors from the plasma membrane and increased the tyrosine kinase activity of p56lck. The anti-CD2 mAb combination (T11(2) + T11(3)) that produced the most rapid increase in p56lck kinase activity also induced the most rapid delocalization of the kinase. In stimulated cells, both p56lck and CD2 receptors are detected in cytoplasmic vesicles. The internalization of p56lck in endocytic vesicles was established by confocal microscopy. By double staining it was shown that only part of the p56lck colocalized with the internalized CD2 receptor suggesting distinct sorting processes. Internalization of p56lck appeared to be specific of CD2 stimulation as: 1) in Jurkat cells triggered with an anti-CD3 mAb, p56lck was not internalized whereas CD3 receptors were completely endocytosed; 2) when cells were stimulated via CD4, the kinase and CD4 receptors remained associated with the plasma membrane. In addition, internalization of p56lck upon stimulation of CD2 receptors was not modified in CD2+/CD3-Jurkat cells indicating that CD3 is not involved in this process. The identification of different subcellular localizations of p56lck in resting and stimulated T cells should represent an important step in the definition of its functional activity.

Antigens, Differentiation, T-Lymphocyte↗