HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction

Nef is an HIV-1 virulence factor that promotes viral pathogenicity by altering host cell signaling pathways. Nef binds several members of the Src kinase family, and these interactions have been implicated in the pathogenesis of HIV/AIDS. However, the direct effect of Nef interaction on Src family kinase (SFK) regulation and activity has not been systematically addressed. We explored this issue using Saccharomyces cerevisiae, a well defined model system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk suppressed SFK activity and reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and c-Src but did not detectably affect Fgr, Fyn, Lck, or Yes. Mutagenesis of the Nef PXXP motif essential for SH3 domain binding greatly reduced the effect of Nef on Hck, Lyn, and c-Src, suggesting that Nef activates these Src family members through allosteric displacement of intramolecular SH3-linker interactions. These data show that Nef selectively activates Hck, Lyn, and c-Src among SFKs, identifying these kinases as proximal effectors of Nef signaling and potential targets for anti-HIV drug discovery.     

Src family kinase activity is required for Kit-mediated mitogen-activated protein (MAP) kinase activation, however loss of functional retinoblastoma protein makes MAP kinase activation unnecessary for growth of small cell lung cancer cells.

Small cell lung cancer (SCLC) is characterized by multiple genetic alterations that include inactivation of the retinoblastoma protein (Rb), the establishment of several autocrine loops including that induced by coexpression of stem cell factor (SCF) and Kit, and the ectopic expression and activation of Src family kinases. Previous studies have shown that Lck associates with, and becomes activated by, Kit after SCF stimulation of SCLC cells. In the present study, we have demonstrated that PP1, a pharmacological inhibitor of Src kinases, blocked SCF-mediated activation of mitogen-activated protein (MAP) kinase, but it also inhibited Kit activation. However, MAP kinase activation was more sensitive than Kit activation to the effects of PP1. Overexpression of Lck reduced the sensitivity of MAP kinase activation to PP1 without altering the sensitivity of Kit activation, which suggested a role for Lck in SCF-mediated MAP kinase activation. Inducible expression of a dominant negative Lck inhibited MAP kinase activation in a dose-dependent manner, which confirmed that Src family kinase activity is required for SCF-induced MAP kinase activation. The growth of cells that expressed dominant negative Lck was unaffected, however, despite the inhibition of MAP kinase. Growth was also unaffected by the inhibition of the MAP kinase pathway using PD 98059, but sensitivity to the MAP/extracellular signal-regulated kinase kinase inhibitor could be partially restored by expression of wild-type Rb. Therefore, MAP kinase activation seems to be dispensable for the growth of SCLC only in the absence of Rb expression. These data suggest that the SCF/Kit autocrine loop, through activation of Lck and subsequently MAP kinase, and the mutational inactivation of Rb contribute to the loss of G1-S phase checkpoint regulation during the pathogenesis of SCLC. Furthermore, the data demonstrate that, in established SCLC cell lines, proliferative signal transduction initiated by Kit is mediated by pathways other than the classic MAP kinase pathway.     

GRIM-1, a novel growth suppressor, inhibits rRNA maturation by suppressing small nucleolar RNAs

We have recently isolated novel IFN-inducible gene, Gene associated with Retinoid-Interferon-induced Mortality-1 (GRIM-1), using a genetic technique. Moderate ectopic expression of GRIM-1 caused growth inhibition and sensitized cells to retinoic acid (RA)/IFN-induced cell death while high expression caused apoptosis. GRIM-1 depletion, using RNAi, conferred a growth advantage. Three protein isoforms (1α, 1β and 1γ) with identical C-termini are produced from GRIM-1 mRNA. We show that GRIM-1 isoforms interact with NAF1 and DKC1, two essential proteins required for box H/ACA sno/sca RNP biogenesis and suppresses box H/ACA RNA levels in mammalian cells by delocalizing NAF1. Suppression of these small RNAs manifests as inefficient rRNA maturation and growth suppression. Interestingly, yeast Shq1p also caused growth suppression in mammalian cells. Consistent with its growth-suppressive property, GRIM-1 expression is lost in a number of human primary prostate tumors. Our observations support a recent study that GRIM-1 might act as a co-tumor suppressor in the prostate.     

Interaction of SAP-1, a transmembrane-type protein-tyrosine phosphatase,with the tyrosine kinase Lck. Roles in regulation of T cell function

SAP-1 is a transmembrane-type protein-tyrosine phosphatase that is expressed in most tissues but whose physiological functions remain unknown. The cytoplasmic region of SAP-1 has now been shown to bind directly the tyrosine kinase Lck. Overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of SAP-1, inhibited both the basal and the T cell antigen receptor (TCR)-stimulated activity of Lck in human Jurkat T cell lines. Lck served as a direct substrate for dephosphorylation by SAP-1 in vitro. Overexpression of wild-type SAP-1 in Jurkat cells also: (i) inhibited both the activation of mitogen-activated protein kinase and the increase in cell surface expression of CD69 induced by TCR stimulation; (ii) reduced the extent of the TCR-induced increase in the tyrosine phosphorylation of ZAP-70 or that of LAT; (iii) reduced both the basal level of tyrosine phosphorylation of p62dok, as well as the increase in the phosphorylation of this protein induced by CD2 stimulation; and (iv) inhibited cell migration. These results thus suggest that the direct interaction of SAP-1 with Lck results in inhibition of the kinase activity of the latter and a consequent negative regulation of T cell function.     

Lck dephosphorylation at Tyr-394 and inhibition of T cell antigen receptor signaling by Yersinia phosphatase YopH

A key virulence factor for Yersinia pestis, the etiologic agent of plague, is the tyrosine phosphatase YopH, which the bacterium injects into host cells. We report that treatment of human T lymphocytes with a recombinant membrane-permeable YopH resulted in severe reduction in intracellular tyrosine phosphorylation and inhibition of T cell activation. The primary signal transducer for the T cell antigen receptor, the Lck tyrosine kinase, was specifically precipitated by a substrate-trapping YopH mutant, and Lck was dephosphorylated at its positive regulatory site, Tyr-394, in cells containing active YopH. By turning off Lck, YopH blocks T cell antigen receptor signaling at its very first step, effectively preventing the development of a protective immune response against this lethal bacterium.     

Superantigen stimulation reveals the contribution of Lck to negative regulation of T cell activation

The conventional paradigm of T cell activation through the TCR states that Lck plays a critical activating role in this signaling process. However, the T cell response to bacterial superantigens does not require Lck. In this study we report that not only is Lck dispensable for T cell activation by superantigens, but it actively inhibits this signaling pathway. Disruption of Lck function, either by repression of Lck gene expression or by selective pharmacologic inhibitors of Lck, led to increased IL-2 production in response to superantigen stimulation. This negative regulatory effect of Lck on superantigen-induced T cell responses required the kinase activity of Lck and correlated with early TCR signaling, but was independent of immunological synapse formation and TCR internalization. Our data demonstrate that the multistage role of Lck in T cell signaling includes the activation of a negative regulatory pathway of T cell activation.     

Heparin down-regulates the phorbol ester-induced protein kinase C gene expression in human endothelial cells: enzyme-mediated autoregulation of protein kinase C-alpha and -delta genes.

Overexpression of protein kinase C-alpha and protein kinase C-delta has been shown to modulate a number of biological effects, including the cell growth and differentiation. We hypothesized that heparin, a potent antimitogenic drug, could affect the cell proliferation by inhibiting the expression of specific protein kinase C genes. Heparin, markedly but not completely, inhibited the serum-stimulated protein kinase C-alpha and -delta mRNA expression. Protein kinase C inhibition or down-regulation significantly decreased the serum-induced protein kinase C isoenzyme gene expression. Heparin failed to inhibit the residual effect of serum that was resistant to the above-mentioned treatments. Phorbol 12-myristate 13-acetate elicited an increase of protein kinase C isoenzyme gene expression that was completely prevented by protein kinase C inhibition or down-regulation. Heparin dose-dependently counteracted and ultimately abolished the increase in the protein kinase C isoenzyme gene expression elicited by phorbol 12-myristate 13-acetate. These results suggest that the inhibition of an autoregulatory role wielded by protein kinase C on the protein kinase C-alpha and -delta gene expression might represent a possible mechanism by which glycosaminoglycans modulate the cell growth.     

LckBP1, a proline-rich protein expressed in haematopoietic lineage cells, directly associates with the SH3 domain of protein tyrosine kinase p56lck.

The Lck tyrosine kinase molecule plays an essential role in T cell activation and T cell development. Using the expression cloning technique, we have isolated a gene that encodes a molecule, LckBP1, able to associate with murine Lck. Analysis of full-length LckBP1 cDNA indicates at least four potentially important segments: a four tandem 37 amino acid repeat motif with a potential helix-turn-helix DNA binding motif; a proline-rich region; a proline-glutamate repeat; and an SH3 domain. These four regions are very similar to the human haematopoietic-specific protein 1 (HS1). Deletion mutant analysis of LckBP1 revealed two proline-rich regions that permit association with Lck SH3. One region contains prolines conserved among HS1 and cortactin, and the other region contains a potential MAP kinase recognition site. In vivo association between Lck and LckBP1 was confirmed by immunoprecipitation of lysates from a pre-T cell line and adult thymocytes using antibodies specific for Lck and LckBP1. LckBP1 is tyrosine phosphorylated after T-cell receptor stimulation. The SH3 domain and the potential helix-turn-helix motif in LckBP1 suggest that this molecule may associate with various molecules and function as a DNA binding molecule. The data also suggest that LckBP1 mediates intracellular signalling through Lck in T cells.     

Yeast GMP kinase mutants constitutively express AMP biosynthesis genes by phenocopying a hypoxanthine-guanine phosphoribosyltransferase defect

We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes). We show that bra3 mutations are alleles of the GUK1 gene, which encodes GMP kinase. The bra3 mutants have a low GMP kinase activity, excrete purines in the medium, and show vegetative growth defects and resistance to purine base analogs. The bra3 locus also corresponds to the previously described pur5 locus. Several lines of evidence indicate that the decrease in GMP kinase activity in the bra3 mutants results in GMP accumulation and feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), encoded by the HPT1 gene. First, guk1 and hpt1 mutants share several phenotypes, such as adenine derepression, purine excretion, and 8-azaguanine resistance. Second, overexpression of HPT1 allows suppression of the deregulated phenotype of the guk1 mutants. Third, we show that purified yeast HGPRT is inhibited by GMP in vitro. Finally, incorporation of hypoxanthine into nucleotides is similarly diminished in hpt1 and guk1 mutants in vivo. We conclude that the decrease in GMP kinase activity in the guk1 mutants results in deregulation of the ADE gene expression by phenocopying a defect in HGPRT. The possible occurrence of a similar phenomenon in humans is discussed.     

Carbon source-dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae

Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells. Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae. With this objective in mind, we transfected S. cerevisiae cells with a plasmid (pYECepsilon) coding for the expression of murine protein kinase C epsilon (PKCepsilon) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECepsilon expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCepsilon antibody. Extracts from these pYECepsilon-transfected cells could phosphorylate a PKCepsilon substrate peptide in a phospholipid/phorbol ester-dependent manner. Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCepsilon was fused in frame with GST (GST-Repsilon), further confirming the successful expression of murine PKCepsilon. Induction of PKCepsilon expression by galactose in cells transformed with pYECepsilon increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol. However, when glucose was used as the carbon source, PKCepsilon expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCalpha or PKCbeta-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively. Visualization of the cells by phase contrast microscopy indicated that murine PKCepsilon expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds. Since overall growth of the cells was dramatically decreased, the data suggests that PKCepsilon expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCepsilon-expressing yeast cells (1-2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKCepsilon expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCepsilon expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle. This murine PKCepsilon-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells.     

Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway

The role of Lck in IL-2-induced proliferation and cell survival is still controversial. Here, we show that the Src family kinase inhibitor, PP1, reduced the IL-2-induced proliferation of human T cells significantly without inhibiting the anti-apoptotic effect of IL-2. As Lck is the only Src family kinase activated upon IL-2 stimulation in T cells, this indicates that Lck is involved in IL-2-induced proliferation but not survival. IL-2-induced MAP kinase activation was only slightly inhibited by PP1, suggesting that Lck is not essential for IL-2-induced MAP kinase activation in human T cells. We found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis fungoides tumor T cells, but Lck had no influence on the delayed kinetics of MAP kinase activation, indicating that Lck is not essential for MAP kinase activation in mycosis fungoides tumor T cells or in non-cancerous T cells. Taken together, this indicates that Lck is involved in IL-2-induced proliferation, but not cell survival, through a pathway not involving MAP kinase.     

Thalidomide suppresses NF-kappa B activation induced by TNF and H2O2, but not that activated by ceramide, lipopolysaccharides, or phorbol ester

Thalidomide ([+]-alpha-phthalimidoglutarimide), a psychoactive drug that readily crosses the blood-brain barrier, has been shown to exhibit anti-inflammatory, antiangiogenic, and immunosuppressive properties through a mechanism that is not fully established. Due to the central role of NF-kappaB in these responses, we postulated that thalidomide mediates its effects through suppression of NF-kappaB activation. We investigated the effects of thalidomide on NF-kappaB activation induced by various inflammatory agents in Jurkat cells. The treatment of these cells with thalidomide suppressed TNF-induced NF-kappaB activation, with optimum effect occurring at 50 microg/ml thalidomide. These effects were not restricted to T cells, as other hematopoietic and epithelial cell types were also inhibited. Thalidomide suppressed H(2)O(2)-induced NF-kappaB activation but had no effect on NF-kappaB activation induced by PMA, LPS, okadaic acid, or ceramide, suggesting selectivity in suppression of NF-kappaB. The suppression of TNF-induced NF-kappaB activation by thalidomide correlated with partial inhibition of TNF-induced degradation of an inhibitory subunit of NF-kappaB (IkappaBalpha), abrogation of IkappaBalpha kinase activation, and inhibition of NF-kappaB-dependent reporter gene expression. Thalidomide abolished the NF-kappaB-dependent reporter gene expression activated by overexpression of TNFR1, TNFR-associated factor-2, and NF-kappaB-inducing kinase, but not that activated by the p65 subunit of NF-kappaB. Overall, our results clearly demonstrate that thalidomide suppresses NF-kappaB activation specifically induced by TNF and H(2)O(2) and that this may contribute to its role in suppression of proliferation, inflammation, angiogenesis, and the immune system.     

Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids

The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.     

Interferon-alpha induction of STATs1, -3 DNA binding and growth arrest is independent of Lck and active mitogen-activated kinase in T cells

Type I interferons (IFNs) are a family of cytokines that have antiviral and antiproliferative effects. Data regarding the processes by which these cytokines transduce signals from the cell membrane to the nucleus are becoming increasingly complex. The most characterized pathway is via JAK-STAT signaling. Previous studies established a potential role for the Src-family kinase Lck in JAK-STAT signaling. Therefore, this study was designed to analyze the role of Lck in IFN-alpha signaling by using the Jurkat, JCam (an Lck-defective cell line derived from Jurkat), and JCam/Lck (JCam cells with Lck restored). The results show that IFN-alpha can induce MAPK activity, but only in cells containing Lck. Furthermore, STATs1 and -3 are effectively phosphorylated and activated to bind DNA in the absence of Lck expression in IFN-alpha-treated cells. Finally, the results demonstrate that IFN-alpha exerts an antiproliferative effect in all three cell lines. These data indicate that Lck and active MAPK do not affect IFN-alpha-induced growth arrest or induction of STAT1s1 and -3 DNA binding ability.