Analysis of the activity and phosphorylation of the lck protein in lymphoid cells

p56lck, the tyrosine protein kinase encoded by the lck gene, is expressed at a 40-fold elevated level in the LSTRA cell line. This is associated with increased tyrosine phosphorylation of cellular proteins. We have asked here whether the increased tyrosine protein phosphorylation is due to an altered activity of the protein or to the unusually high level of p56lck. In vitro protein kinase assays showed that neither the specific activity nor the affinity of p56lck for two different substrates was abnormal in LSTRA cells. Additionally, analysis of the phosphorylation of p56lck in LSTRA and other cell lines showed that the protein was phosphorylated extensively at a negative-regulatory site, Tyr 505, in all of the cells examined. Since the primary structure of the p56lck expressed at a high level in LSTRA cells is the same as that found in normal thymus and we found no evidence of activation of the protein by dephosphorylation, it appears that high levels of p56lck can induce increased tyrosine protein phosphorylation in lymphoid cells. In contrast, high levels of the closely related protein, p60c-src have no significant effect on tyrosine protein phosphorylation in fibroblasts. The regulation of the protein kinase activity of p56lck in lymphoid cells may therefore differ from the regulation of p60c-src in fibroblasts.     

Functional analysis of the SH2 and SH3 domains of the lck tyrosine protein kinase.

p56lck is a lymphoid cell-specific member of the src family of cytoplasmic tyrosine kinases. In helper and cytotoxic T cells it is physically associated with the CD4 and CD8 surface antigens and appears to play a role in signal transduction during T-cell activation. p56lck contains both an SH3 and an SH2 Src homology domain. Such domains have been suggested to play a role in the regulation of the activity or function of both receptor and non-receptor tyrosine protein kinases. Deletion of either or both domains in p56lck was found here to activate the protein and to lead to increased phosphorylation of the autophosphorylation site, Tyr-394, in vivo. These findings are consistent with the hypothesis that these domains participate in repression of the kinase activity of p56lck. None of the deleted forms was capable of transformation of fibroblasts. Deletion of the SH3 domain of a constitutively activated form of p56lck, p56lckF505, did not diminish the transforming activity of this protein. This suggests that this domain is dispensable for the transformation of fibroblasts by p56lck. In contrast, deletion of the SH2 domain abolished the transforming potential of activated p56lckF505. However, interpretation of this effect is made somewhat difficult because the mutation also lowered the steady-state abundance of the protein.     

Human T-cell leukemia virus type-I-infected T-cell lines scarcely produce p56lck, whether or not they express lck mRNA

We have previously reported that lck mRNA (a lymphocyte-specific protein tyrosine kinase gene) is absent in human T-cell leukemia virus type I (HTLV-I)-infected interleukin-2(IL-2)-independent T-cell lines, while HTLV-I-negative T-cell lines and HTLV-I-positive IL-2-dependent ones express a large amount of lck mRNA. To further investigate the levels of lck expression, we prepared rabbit anti-Lck antiserum directed against the synthetic oligopeptide of 32 amino acids corresponding to the carboxy terminus of this gene product, p56lck. Using this antiserum, we show that HTLV-I-positive T-cell lines, whether they are IL-2-dependent or not, scarcely express p56lck. In other words, IL-2-dependent HTLV-I-positive T-cell lines seldom produce p56lck in spite of high expression of lck mRNA. Absence of p56lck is suspected of playing an important role in malignant transformation of HTLV-I-infected T-cells.     

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.     

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.     

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.     

Accumulation of p60lck in HTLV-I-transformed T cell lines detected by an anti-lck monoclonal antibody, MOL 171

The lck gene encodes a protein tyrosine kinase of nonreceptor type, p56lck, whose expression occurs almost exclusively in T lymphocytes. MOL 171, an anti-p56lck monoclonal antibody, was produced by using a 25-amino-acid synthetic polypeptide as the antigen, its structure corresponding to the N-terminal region deduced from the lck cDNA sequence. Immunoblot analysis with MOL 171 showed the accumulation of 60 kD form of Lck protein, p60lck, and the decrease of p56lck in human T cell leukemia virus type I (HTLV-I)-transformed T cell lines. Another anti-Lck monoclonal antibody, MOL 294, raised by using a synthetic peptide corresponding to the C-terminal region deduced from the lck cDNA sequence, also detected the accumulation of p60lck in those HTLV-I-transformed T cell lines. The appearance of p60lck with the decrease of p56lck in normal T lymphocytes after stimulation suggested the origin of p60lck in HTLV-I-transformed T cells.     

An oncogenic tyrosine kinase inhibits DNA repair and DNA-damage-induced Bcl-xL deamidation in T cell transformation

A transgenic mouse model of T cell lymphoma was used to investigate the transforming events mediated by an oncogenic tyrosine kinase in pretumorigenic CD4-CD8- (DN) thymocytes. Parental CD45(-/-) and p56(lck-F505Y) mice do not develop tumors, whereas their CD45(-/-)p56(lck-F505Y) progeny develop T lymphomas. Increased but nononcogenic p56lck kinase activity in p56(lck-F505Y) mice DN thymocytes causes cell-cycle progression, survival, and Bcl-XL upregulation. Additional unique oncogenic signals occur in pretumorigenic CD45(-/-)p56(lck-F505Y) thymocytes in which p56lck kinase activity is 2- to 3-fold higher relative to p56(lck-F505Y): inhibition of DNA repair, inhibition of DNA-damage-induced Bcl-XL deamidation, Bax conformational change and mitochondrial translocation, cytochrome c release, and the apoptotic caspase execution cascade. Inhibition of Bcl-XL deamidation may be a critical switch in oncogenic kinase-induced T cell transformation.     

Dephosphorylation and activation of the T cell tyrosine kinase pp56lck by the leukocyte common antigen (CD45)

pp56lck, encoded by the lck proto-oncogene, is a non-receptor tyrosine protein kinase (TPK) found in all T lymphocytes. A significant fraction of pp56lck is tightly associated with the cytoplasmic domains of CD4 and CD8 glycoproteins, suggesting an important role for it in thymic development and T cell activation. We have studied the regulation of the tyrosine kinase activity of pp56lck and found recently that it becomes rapidly activated in isolated T cell membranes by an endogenous mechanism apparently involving a phosphotyrosine phosphatase (PTPase). Moreover pp56lck was activated upon addition of isolated CD45 (leukocyte common antigen), a major membrane PTPase, and activation was absent in T cell mutants lacking CD45. Here, we address in more detail the mechanisms of pp56lck activation by the CD45 PTPase and show that this event is time-dependent, sensitive to Na3VO4 (which blocks the PTPase activity of CD45), and is accompanied by dephosphorylation of a regulatory tyrosine residue (Tyr-505) near the C-terminus of pp56lck. This provides a molecular mechanism for regulation of the catalytic activity of pp56lck by CD45, which thus is likely to be the physiological activator of pp56lck. Activation of pp56lck by the CD45 PTPase may underlie many of the reported immunomodulatory effects of antibodies to CD45, and provides further support for the notion that CD45-mediated tyrosine dephosphorylation plays a critical role in T cell activation and growth.     

Inactivation of the p53 oncogene by internal deletion or retroviral integration in erythroleukemic cell lines induced by Friend leukemia virus

The p53 gene is rearranged in a high proportion of erythroleukemic cell lines derived from the spleens of mice infected with Friend leukemia virus. These rearrangements result in either the synthesis of a truncated protein or the inactivation of the p53 gene. Here we have molecularly characterized the rearrangements in two murine erythroleukemic cell lines induced by Friend leukemia virus, DP20-1 and CB3, that contain a rearranged p53 gene and fail to express p53 protein. The rearrangement in the DP20-1 cell line is due to the insertion of Friend spleen focus-forming provirus (SFFV) in the 3' end of the p53 gene in intron sequences between exons 9 and 10. Transfection of molecular clones of this SFFV provirus into NIH3T3 cells results in the generation of infectious virus as determined by its ability, in the presence of helper virus, to induce rapid splenomegaly and polycythemia when injected into adult DBA/2J mice. Insertion of SFFV in DP20-1 cells resulted in the expression of an aberrant 2.9 kb RNA species. Analysis of a molecular clone of the rearranged p53 gene in a second cell line, CB3, revealed that the p53 gene in this clone has sustained a large deletion within the p53 gene resulting in the loss of coding sequences between exons 4 and 8. The 5' end of the deletion originates within exon 4 and extends 3' to within the eighth intron. The significance of these findings with regard to the multi-stage nature of Friend virus induced erythroleukemia is discussed.     

Accumulation of p60lck in HTLV-I-transformed T Cell Lines Detected by an Anti-Lck Monoclonal Antibody, MOL 171

The lck gene encodes a protein tyrosine kinase of nonreceptor type, p56^lck, whose expression occurs almost exclusively in T lymphocytes. MOL 171, an anti-p56^lck monoclonal antibody, was produced by using a 25-amino-acid synthetic polypeptide as the antigen, its structure corresponding to the N-terminal region deduced from the lck cDNA sequence. Immunoblot analysis with MOL 171 showed the accumulation of 60 kD form of Lck protein, p60 ^lck , and the decrease of p56 ^lck in human T cell leukemia virus type I (HTLV-I)-transformed T cell lines. Another anti-Lck monoclonal antibody, MOL 294, raised by using a synthetic peptide corresponding to the C-terminal region deduced from the lck cDNA sequence, also detected the accumulation of p60 ^lck in those HTLV-1-transformed T cell lines. The appearance of p60 ^lck with the decrease of p56 ^lck in normal T lymphocytes after stimulation suggested the origin of p60 ^lck in HTLV-I-transformed T cells.     

Phosphorylation and regulatory effects of the carboxy terminus of a Drosophila src homolog.

The kinase activities of the vertebrate src family members are repressed by phosphorylation of a tyrosine residue in the carboxy-terminal 'tail' of these molecules. To explore whether the tail of an invertebrate src homolog might also serve a regulatory function, we examined the ability of the carboxy terminus of a Drosophila src homolog (p62D), which contains a tyrosine homologous to those in the vertebrate src family members, to regulate the following molecules in mammalian fibroblasts: (1) a chimeric protein, p60CD, containing the amino terminus and catalytic domains of chicken p60c-src joined to the C-terminus of p62D; and (2) full-length p62D itself. By a variety of criteria p60CD appears to be a partially, rather than fully, repressed form of p60c-src. Phosphopeptide mapping indicates that partial repression correlates with partial phosphorylation of the tyrosine in the p62D tail of the chimera. Phosphorylation of the tail may also regulate full-length p62D. Expression of p62D in fibroblasts does not affect cell morphology or the overall abundance of tyrosine-phosphorylated proteins. The molecule is phosphorylated at its C-terminal tyrosine (Tyr-547), but not at its in vitro autophosphorylation sites, suggesting that it is catalytically repressed in fibroblasts. Expression of a truncated p62D mutant lacking Tyr-547 is associated with a clear alteration in cellular morphology and a two- to threefold increase in cellular phosphotyrosine levels. These results suggest that phosphorylation of the C-terminal tyrosine of the tail of an invertebrate src-like kinase can repress the activity of adjacent catalytic domains.     

Transformation of murine pre-B-lymphocytes by v-erb-B: progression to growth factor independence and tumorigenicity.

v-erb-B is the principal transforming gene of avian erythroblastosis virus, a replication defective retrovirus that transforms erythroid and fibroblast cells in vitro and causes erythroleukemia and fibrosarcoma in vivo. We have used a recombinant murine retrovirus, based on the truncated genome of Moloney murine leukemia virus and containing a chimeric gag-v-erb-B gene, to determine the murine hematopoietic cells transformed by v-erb-B. Infection of day 16.5 murine embryonal cells in liquid culture with this virus resulted in the outgrowth of foci of loosely to non-adherent hematopoietic cells which grew in close association with an adherent monolayer. After several weeks in culture a clonal population of transformed pre-B-lymphocytes emerged from this transformation initiated, though still growth factor dependent, population. These transformed cells were growth factor independent, and were tumorigenic in syngeneic mice. The results indicate that although v-erb-B can initiate transformation of murine hematopoietic cells, additional events are required for establishment of the fully transformed growth factor independent, tumorigenic phenotype. This v-erb-B induced progression from growth factor dependence to independence provides an in vitro model system for analysing events involved in the initiation and maintenance of leukemia.     

Transformation of a mouse cell line by murine sarcoma virus (Moloney)

Murine sarcoma virus (Moloney) (MSV) transformed C3H2K cells originating from the kidney tissues of a newborn C3H/He mouse. Titration of the virus showed a one-hit dose response in the presence of an excess of Friend leukemia virus (FLV) and a two-hit dose response in the absence of FLV, indicating that MSV was defective and a dual infection by MSV and a murine leukemia virus was required for MSV focus formation. Preinfection with FLV interfered with focus formation by MSV. The MSV foci appeared most abundantly when the cells in the S phase were infected with the virus. Remarkable similarities between murine and avian sarcoma viruses were thus established. Focus center assay on either uninfected or FLV-infected C3H2K cell monolayer indicated that MSV foci did not arise by cellular division alone but required secondary infection, while RSV foci arose by cellular division alone.     

Localization and modulation of the DNA-binding activity of the human c-ets-1 protooncogene

The avian acute leukemia virus (E26) induces a mixed erythroid-myeloid leukemia in chickens and carries two distinct oncogenes, v-myb and v-ets. The viral protein responsible for transformation is a gag-myb-ets fusion protein that is located in the nucleus of the transformed cells. The cellular homologue of v-ets (c-ets-1) is highly expressed in lymphoid cells and differs from the v-ets gene at its carboxy terminal region. Here, we show that both the c-ets-1 and v-ets gene products are DNA-binding proteins and their DNA-binding activity is located in the carboxy terminal (46 amino acid residues) region. It appears that this DNA-binding activity is modulated by the extreme carboxy terminal region. The amino acid sequences of the putative ets DNA-binding domain at its carboxy terminal region showed a helix-turn-helix secondary structure. Exchanging the nonhomologous extreme carboxy terminal regions of c-ets-1 with v-ets gene sequences showed differences in DNA-binding affinity, indicating that these differences may be partly responsible for the activation of the ets oncogene.     

Non-producer human cells induced by murine sarcoma virus.

Non-produces (NP) human cells were isolated from transformed foci induced by the Kirsten mouse sarcoma virus. These morphologically altered NP cells produced neither infectious virus nor complement-fixing antigens of the murine sarcoma-leukemia virus complex. However, the sarcoma virus genome could be rescued from these NF cells by co-cultivation with cells carrying "helper" Kirsten mouse leukemia virus or Woolly Monkey leukemia virus. The possible usefulness of these cells in efforts designed to detect covert or repressed RNA tumor viruses in various animal and human tissues is discussed.     

Expression of the p56lck by colon tumors: a marker of their invasive capacity?

Since its discovery in murine thymoma in 1982, the p56lck (lymphocyte cellular kinase) has been shown to be a pivotal enzyme to both maturation of thymocytes and activation and proliferation of peripheral lymphocytes. The p56lck sequence appeared highly homologous to that of the oncogene p60c-src as did its exon-intron organisation. These data have suggested the lck gene originates from the ancestral src gene by the exon-shuffling mechanism. However, and in spite of this relationship with the p60src oncogene which is often implicated in human cancers, p56lck does not appear involved in lymphoproliferative diseases, either by overexpression or activating mutations. Nevertheless, its aberrant expression has been reported in some carcinomas (colon, lung and mammary). This unexpected expression of a lymphoid-specific protein in solid tumors remained enigmatic until recent studies. In this review, we report these data and explain the possible mechanisms which could lead to the p56lck ectopic expression. We also discuss of signalling pathways which could be affected by the abnormal presence of the p56lck in these tumoral epithelial cells. In particular, we indicate that p56lck could favor metastases by facilitating loss of cell adhesion.