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Biomedical subjects

A Veillette

Publications and source records attributed to A Veillette.

At least 73 records · Page 4Linked to original sources
PubMed1992

Src-related protein tyrosine kinases and T-cell receptor signalling.

Upon antigen stimulation, the T-cell receptor for antigen transduces an intracellular protein tyrosine phosphorylation signal that is critical for subsequent T-lymphocyte activation. As the antigen receptor does not possess an intrinsic protein tyrosine kinase activity, the mechanism by which it regulates protein tyrosine phosphorylation is unconventional. Evidence is increasing that the Src-related protein tyrosine kinases P56lck and p59fyn, as well as the protein tyrosine phosphatase CD45, are involved in this process.

Genes, src↗
PubMed1992

tkl is the avian homolog of the mammalian lck tyrosine protein kinase gene.

We have tested the possibility that tkl, a partially characterized avian tyrosine protein kinase gene, is the chicken homolog of lck, a lymphocyte-specific mammalian gene. Using polymerase chain reactions, we have cloned sequences encoding the previously unidentified amino terminus of the tkl gene product. The newly defined unique domain of Tkl displayed significant identity (68%) to the equivalent region of the mammalian lck gene product, p56lck. This identity included a glycine residue at position 2 (present in all Scr-related tyrosine protein kinases) and a cysteine motif at positions 20 and 23, which allows binding of p56lck to CD4 and CD8 in mammalian T lymphocytes. A specific RNase protection assay revealed that, in contrast to a previous report (K. Strebhardt, J. I. Mullins, C. Bruck, and H. Rübsamen-Waigmann, Proc. Natl. Acad. Sci. USA 84:8778-8782, 1987), tkl expression is restricted to the lymphoid tissues thymus and spleen. Moreover, the absence of tkl transcripts in the bursa of Fabricius suggested that this gene is expressed in avian T lymphocytes but not in B lymphocytes. A polyclonal rabbit antiserum raised against the unique domain of Tkl recognized a 56-kDa polypeptide with associated protein kinase activity from avian thymus-derived cells. Additional studies showed that p56tkl is structurally similar to mammalian p56lck and that it is physically associated with the avian CD4 and CD8 T-cell surface antigens. It was also determined that tkl transcripts have one major type of 5' untranslated region (UTR), which differs greatly from the two known 5' UTRs of mammalian lck mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.

Animals↗
PubMed1992

Regulation of the enzymatic function of the lymphocyte-specific tyrosine protein kinase p56lck by the non-catalytic SH2 and SH3 domains.

The enzymatic activity of the lymphocyte-specific tyrosine protein kinase p56lck appears to be tightly regulated by phosphorylation of the conserved carboxy-terminal tyrosine residue 505. Indeed, substitution of this tyrosine residue by a non-phosphorylatable phenylalanine results in a constitutively activated version of p56lck that can transform rodent fibroblasts. In this report, we evaluate the functions of the conserved non-catalytic Src homology (SH) domains 2 and 3 of p56lck in the regulation of its enzymatic activity in NIH3T3 fibroblasts. We found that deletion of the SH2 or, to a lesser extent, the SH3 domain of p56lck resulted in an increase in the tyrosine protein kinase activity of wild-type Lck polypeptides. The SH2 domain (but not the SH3 domain) was also required for full oncogenic transformation by Lck molecules activated through removal of tyrosine 505. This effect did not appear to be the result of a diminution of the enhanced catalytic activity of F505 Lck polypeptides. However, it may relate to the findings that the SH2 domain can bind and possibly enhance phosphorylation of specific phosphotyrosine-containing proteins. Taken together, these observations imply roles for the non-catalytic SH2 and SH3 domains in the regulation of the catalytic activity of p56lck. They suggest that the enzymatic function of this Src-related polypeptide is physiologically repressed by processes dependent on the presence of the SH2 and SH3 sequences. Moreover, they indicate that the SH2 domain also plays a positive role in the function of activated p56lck molecules in NIH3T3 cells.

Animals↗
PubMed1991

Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

Lymphocyte-specific tyrosine protein kinase p56lck is physically associated with CD4 and CD8 T-cell surface molecules, suggesting that it may transduce CD4/CD8-triggered tyrosine phosphorylation signals during antigen stimulation. Indeed, antibody-mediated aggregation of CD4 (to mimic interaction with its ligand, major histocompatibility complex (MHC) class II molecules), rapidly elevates the kinase activity of p56lck and is associated with marked changes in tyrosine protein phosphorylation. Genetic analyses suggest that the interaction of CD4/CD8 with p56lck results in a positive signal during antigen-induced T-cell activation. To evaluate directly the role of p56lck in T-cell activation, we introduced a constitutively activated form of Lck protein (tyrosine 505 to phenylalanine 505 mutant); in a CD4-negative, MHC-class II restricted mouse T-cell hybridoma. We report here that, as for transfection of CD4, expression of the Lck mutant enhanced T-lymphocyte responsiveness. This finding provides direct evidence that p56lck can positively regulate T-cell functions and that it mediates at least some of the effects of CD4 and CD8 on T-cell activation.

Animals↗
PubMed1991

Avian CD4 and CD8 interact with a cellular tyrosine protein kinase homologous to mammalian p56lck.

Functional as well as immunochemical studies have indicated that chicken T lymphocytes express surface antigens homologous to mammalian CD4 and CD8. The results presented in this report demonstrate that the avian CD4 and CD8 homologs are physically associated with a cellular tyrosine protein kinase related to mammalian p56lck. Furthermore, they show that engagement of either avian CD4 or CD8 is associated with a rapid intracellular tyrosine phosphorylation signal, suggesting that the function of these surface molecules may be mediated through alterations of the associated tyrosine protein kinase. Taken together, these findings demonstrate that the associations of CD4 and CD8 with an intracellular tyrosine protein kinase are evolutionarily conserved, further enhancing the likelihood that they are critical for normal immune functions.

Animals↗
PubMed1991

Tyrosine phosphorylation of GAP and GAP-associated proteins in lymphoid and fibroblast cells expressing lck.

The Ras GTPase activating protein (GAP) is a strong candidate for the protein that links protein-tyrosine kinases to the Ras mitogenic pathway. GAP and two associated proteins, p62 and p190, were shown to be phosphorylated on tyrosine in the LSTRA thymoma cell line, in which the p56lck tyrosine kinase is overexpressed as a result of retroviral promoter insertion. In NIH3T3 fibroblasts expressing specific oncogenic and transformation-defective variants of p56lck, we found that the tyrosine phosphorylation of GAP complexes required both enzymatic activation and myristylation of p56lck, and correlated with lck transforming activity. The interaction between p62 and p190 from lck-transformed fibroblasts and GAP could be reconstituted in vitro using bacterial TrpE fusion proteins containing GAP Src homology 2 (SH2) domains. In vitro complex formation was insensitive to the prior denaturation of SH2 ligands, suggesting that SH2-binding sites are formed by linear peptide sequences. These results suggest that the tyrosine phosphorylation of GAP, and its interactions with SH2-binding proteins, may be involved in fibroblast transformation by activated lck, and may participate in signal transduction and cellular transformation in lymphoid cells.

Animals↗
PubMed1991

The lymphocyte-specific tyrosine protein kinase p56lck.

The CD4 and CD8 T cell surface antigens are physically associated with the tyrosine protein kinase p56lck. Accumulating data indicate that p56lck transduces intracellular tyrosine protein phosphorylation signals upon engagement of CD4 and CD8 by major histocompatibility complex (MHC) determinants expressed on antigen-presenting cells (APCs). Recent studies show that these p56lck-related phosphorylation events enhance T cell receptor (TCR)-mediated functions and are critical for the proposed co-receptor roles of CD4 and CD8. p56lck is also capable of enhancing antigen receptor responsiveness in the absence of CD4 or CD8 expression, suggesting that it can directly contribute to the TCR-induced tyrosine phosphorylation signal.

Amino Acid Sequence↗
PubMed1990

The cytoplasmic domain of CD4 is required for stable association with the lymphocyte-specific tyrosine protein kinase p56lck.

The CD4 T cell surface molecule binds MHC class II determinants expressed on antigen-presenting cells. CD4 is thought to enhance T cell activation by serving as an adhesion molecule as well as possibly by transducing an independent intracellular signal during the process of antigen stimulation. The recent observation that CD4 is physically associated with the Src-related tyrosine protein kinase p56lck suggests that tyrosine phosphorylation might be involved in these CD4 "signaling" events. The results presented in this report demonstrate that deletion of the cytoplasmic domain of CD4 significantly diminishes its ability to stably associate with p56lck. This observation provides a biochemical basis for the decreased ability of this mutant CD4 molecule to enhance T cell activation during suboptimal antigen stimulation.

Amino Acid Sequence↗
PubMed1990

Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation.

Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.

Animals↗
PubMed1990

The CD4 associated tyrosine protein kinase p56lck is positively regulated through its site of autophosphorylation.

Antibody-mediated aggregation of the CD4 T-cell surface antigen activates bound p56lck molecules and can result in increased intracellular tyrosine protein phosphorylation. To evaluate the basis of the CD4 induced tyrosine phosphorylation signal, we have studied the ability of CD4 to regulate the function of p56lck when these two molecules are co-expressed in non-lymphoid cells. Our studies indicate that cross-linking of CD4 is capable of activation of p56lck in fibroblasts in a manner analogous to that previously reported for T-lymphocytes. They also demonstrate that replacement of the major site of autophosphorylation of p56lck (tyrosine 394) by a phenylalanine residue abolishes the ability to activate p56lck by CD4 cross-linking, implying that this residue is critical for the positive regulation of the Lck tyrosine kinase activity by CD4. Contrary to what we have previously reported for an antigen-dependent murine T-cell clone, as well as murine thymocytes, the CD4 induced activation of p56lck observed in fibroblasts does not result in marked changes in Lck tyrosine phosphorylation, suggesting that other lymphoid specific components may be required for these tyrosine phosphorylation changes.

Animals↗
PubMed1989

Inability of CD8 alpha' polypeptides to associate with p56lck correlates with impaired function in vitro and lack of expression in vivo.

T-cell accessory molecules, particularly CD4 and CD8, seem to be involved in the control of T-cell activation by antigen. Precisely how such molecules operate is not fully understood, but evidence to date suggests a dual role, as receptors binding ligands on stimulator cells and by direct or indirect involvement in intracellular signalling events. In mouse, truncated 'tailless' CD8 molecules occur naturally (CD8 alpha' polypeptides) and although they are expressed on the surface of thymocytes, they are not expressed on the surface of mature T cells. In this study, we show that truncated CD8 molecules are impaired in their ability to interact with the protein tyrosine kinase, p56lck, and have decreased ability to restore immune responsiveness in vitro. Our data support a dual function for CD8 molecules correlated with expression of external domains and cytoplasmic domains, respectively. Both functions appear to be critical for a competent immune system in vivo.

Animals↗
PubMed1989

Engagement of CD4 and CD8 expressed on immature thymocytes induces activation of intracellular tyrosine phosphorylation pathways.

Accumulating data suggest that the target cells for selection events leading to establishment of the mature T cell repertoire are the functionally immature double-positive (CD4+/CD8+) thymocytes, and that the CD4 and CD8 antigens expressed on these cells play important roles in these processes. In an attempt to define the biochemical pathways implicated in these events, we have studied the effects of engagement of accessory molecules on tyrosine protein phosphorylation. The results of our experiments demonstrate that engagement of CD4 and CD8 expressed on double-positive thymocytes is coupled with a rapid tyrosine protein phosphorylation signal. Further analyses have revealed that these two surface molecules are physically associated with the internal membrane tyrosine protein kinase p56lck in immature thymocytes, and that the catalytic function of lck expressed in double-positive thymocytes is significantly enhanced upon engagement of CD4. These data provide evidence that tyrosine-specific protein phosphorylation pathways coupled to the CD4 and CD8 T cell surface antigens are functional in immature thymocytes, and therefore, formally prove that signaling functions of CD4 and CD8 molecules are operative in immature thymocytes.

Animals↗
PubMed1989

Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck.

The CD4 T-cell surface antigen is an integral membrane glycoprotein of relative molecular mass 55,000 which binds class II major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs). It is thought to stabilize physical interactions between T cells and APCs (for a review, see ref. 1). Evidence is accumulating that suggests that CD4 can transduce an independent signal during T-cell activation. It has recently been shown that CD4 expressed on human and murine T cells is physically associated with the Src-related tyrosine protein kinase p56lck (refs 7, 8). These results indicate that CD4 can function as a signal transducer and suggest that tyrosine phosphorylation events may be important in CD4-mediated signalling. Here, we present evidence that cross-linking of the CD4 receptor induces a rapid increase in the tyrosine-specific protein kinase activity of p56lck and is associated with the rapid phosphorylation of one of the subunits (zeta) of the T-cell receptor complex on tyrosine residues. These data provide direct evidence for a specific CD4 signal transduction pathway that is mediated through p56lck and suggest that some of the tyrosine phosphorylation events detected during antigen-mediated T-cell activation may result from signalling through this surface molecule.

Animals↗
PubMed1989

A function for the lck proto-oncogene.

Proto-oncogenes encode products that comprise a select group of cellular regulatory proteins whose mutation or aberrant expression can result in oncogenic transformation. With the exception of certain growth factors and their receptors, the definition of normal functions for most proto-oncogene products has been elusive. The discovery that a member of the src-family of tyrosine protein kinases (p56lck) is associated with both the CD4 and CD8 T-lymphocyte surface glycoproteins provides the first clue to understanding the potential physiological functions of this family of proto-oncogenes.

Amino Acid Sequence↗
PubMed1989

Alterations in tyrosine protein phosphorylation induced by antibody-mediated cross-linking of the CD4 receptor of T lymphocytes.

Accumulating data suggest that the CD4 T-cell surface antigen transduces an independent intracellular signal during antigen-mediated T-cell activation. CD4 is physically associated with the internal membrane tyrosine protein kinase p56lck and can mediate, after antibody-mediated cross-linking, the rapid enzymatic activation of Lck, implying that CD4 signalling may involve changes in tyrosine protein phosphorylation. In this report, we describe that cross-linking of CD4 results in a series of rapid changes in intracellular tyrosine protein phosphorylation. The most prominent CD4-induced tyrosine phosphorylation change involved p56lck, which became extensively phosphorylated on the carboxy-terminal tyrosine residue 505 and, to a lesser extent, lymphocytes can transduce an intracellular signal resulting in tyrosine protein phosphorylation and strongly suggest that this property of CD4 is mediated through p56lck.

Animals↗