Inhibition of neuropeptide-stimulated tyrosine phosphorylation and tyrosine kinase activity stimulates apoptosis in small cell lung cancer cells

Small cell lung cancer (SCLC) cell growth is sustained by multiple autocrine and paracrine growth loops involving neuropeptides. The bombesin family of peptides are autocrine growth factors in H345 SCLC cells and provide a paradigm for the study of growth factors and mitogenic signaling in SCLC cells. We show that bombesin (and other neuropeptides) stimulates protein tyrosine phosphorylation (particularly focal adhesion kinase) and protein tyrosine kinase (PTK) activity in intact SCLC cells. Furthermore, the broad spectrum neuropeptide receptor antagonist [D-Arg, D = Phe, D-Trp, Leu11]substance P inhibits all neuropeptide-mediated signals (including PTK activation), SCLC cell growth in vivo and in vitro, and also increases the natural rate of apoptosis seen in growing SCLC cell lines. Hence the effect of selective PTK inhibition on SCLC cell growth and apoptosis was examined. We show that selective inhibition of PTK activity, with genistein and (3,4,5-tri-hydroxyphenyl)-methylene(-propanedinitrile) tyrphostin-25 inhibits basal and neuropeptide-stimulated SCLC cell growth. Genistein and tyrphostin-25 also stimulate apoptosis in SCLC cells. Inhibition of proliferation in these cells is intimately linke to apoptosis, because these changes occurred without any effect on SCLC cell cycle kinetics, suggesting that apoptosis occurs independently of the cell cycle and that failure to progress through the cell cycle results in apoptosis. Because tyrphostin-25 fails to influence p53 or Bcl-2 expression in these cells, this mode of programmed cell death appears to be via a p53- and Bcl-2-independent mechanism. These results provide evidence that tyrosine phosphorylation is a mitogenic signal in SCLC cells and suggest that regulation of the level of protein tyrosine phosphorylation represents a critical determinant of whether SCLC cells survive and proliferate or die by apoptosis. Thus PTK inhibition may provide a novel therapeutic option in SCLC that has become resistant to conventional chemotherapeutic agents.     

Cell growth inhibition by a novel vitamin K is associated with induction of protein tyrosine phosphorylation

We have shown that a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), potently inhibits cell growth and suggested that the analog exerts its effects mainly via sulfhydryl arylation rather than redox cycling. Since protein-tyrosine phosphatases (PTPases), which have pivotal roles in many cellular functions, have a critical cysteine in their active site, we have proposed PTPases as likely targets for Cpd 5. To test this hypothesis, we examined the effects of Cpd 5 on protein tyrosine phosphorylation of cellular proteins and on the activity of PTPases. We found that Cpd 5 rapidly induced protein tyrosine phosphorylation in a human hepatocellular carcinoma cell line (Hep3B) at growth inhibitory doses, and the effect was blocked by thiols but not by non-thiol antioxidants or tyrosine kinase inhibitors. Cpd 5 inhibited PTPase activity, which was also significantly antagonized by reduced glutathione. Furthermore, the well studied PTPase inhibitor orthovanadate also induced protein tyrosine phosphorylation and growth inhibition in Hep3B cells. These results suggest that inhibition of cellular PTPases by sulfhydryl arylation and subsequent perturbation of protein tyrosine phosphorylation may be involved in the mechanisms of Cpd 5-induced cell growth inhibition.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.     

Normal Syk protein level but abnormal tyrosine phosphorylation in B-CLL cells

One characteristic of B cells that accumulate during chronic lymphocytic leukemia (CLL) is their highly heterogeneous functional responses to B cell receptor (BCR) stimulation. Leukemic B cells with very poor responses have defective rapid tyrosine phosphorylation of numerous substrates, especially phospholipase C (PLC)gamma, as well as a defective calcium elevation on BCR stimulation. This points to a defect in BCR-associated protein tyrosine kinase (PTK). We investigated whether a defect in Syk, a PTK that is pivotal in coupling BCR to downstream signaling events, could account for these alterations. Syk tyrosine phosphorylation triggered by BCR ligation was severely impaired in B-CLL cells with low calcium responses to anti-mu stimulation. Syk associations were also defective, as concomitant tyrosine phosphorylation of a Syk-associated 145 kDa protein comigrating with PLCgamma-2 was only detected in responding B-CLL cells. By contrast, we found similar expression of the kinase regardless of B-CLL cell responsiveness. These results are consistent with the possibility that very proximal BCR signaling elements in some B-CLL cells are unable to connect with downstream biochemical events dominated by tyrosine phosphorylation and the potential docking function of Syk PTK.     

Protein tyrosine phosphatase inhibitors in Fc gamma RI-induced myeloid oxidant signaling

Fc-receptor stimulation in myeloid cells results in increased oxygen consumption, termed the respiratory burst, which is coupled to a rapid and transient increase in tyrosine phosphorylation of cellular proteins. In a previous paper in this journal we showed that the protein tyrosine phosphatase (PTPase) inhibitors sodium orthovanadate and phenylarsine oxide (PAO) block the Fc gamma RI-induced respiratory burst in interferon-gamma-differentiated U937 cells (U937IF) while augmenting the Fc gamma RI-induced tyrosine phosphorylation of cellular proteins. Herein we examine the effects of PTPase inhibitors on specific molecules involved in Fc gamma RI signaling. We show that orthovanadate and PAO augmented the Fc gamma RI-induced tyrosine phosphorylation of the adaptor protein CBL. CBL interactions with other phosphoproteins, among them SHC and CRKL, were also augmented in response to pretreatment with the PTPase inhibitors. SHC was tyrosine phosphorylated in response to Fc gamma RI stimulation of U937IF cells and bound to the SH2 domain of GRB2 in a stimulation-dependent manner. In fusion protein pull-down experiments the interaction of SHC with the SH2 domain of GRB2 was increased in PTPase inhibitor pretreated U937IF cells in response to Fc gamma RI stimulation. Our data support the hypothesis that a tyrosine dephosphorylation event is required for effective transmission of the Fc gamma RI signal to result in activation of the myeloid respiratory burst response.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

Modulation of rod photoreceptor cyclic nucleotide-gated channels by tyrosine phosphorylation

Cyclic nucleotide-gated (CNG) channels in vertebrate photoreceptors are crucial for transducing light-induced changes in cGMP concentration into electrical signals. In this study, we show that both native and exogenously expressed CNG channels from rods are modulated by tyrosine phosphorylation. The cGMP sensitivity of CNG channels, composed of rod alpha-subunits expressed in Xenopus oocytes, gradually increases after excision of inside-out patches from the oocyte membrane. This increase in sensitivity is inhibited by a protein tyrosine phosphatase (PTP) inhibitor and is unaffected by three different Ser/Thr phosphatase inhibitors. Moreover, it is suppressed or reversed by application of ATP but not by a nonhydrolyzable ATP analog. Application of protein tyrosine kinase (PTK) inhibitors causes an increase in cGMP sensitivity, but only in the presence of ATP. Taken together, these results suggest that CNG channels expressed in oocytes are associated with active PTK(s) and PTP(s) that regulate their cGMP sensitivity by changing phosphorylation state. The cGMP sensitivity of native CNG channels from salamander rod outer segments also increases and decreases after incubation with inhibitors of PTP(s) and PTK(s), respectively. These results suggest that rod CNG channels are modulated by tyrosine phosphorylation, which may function as a novel mechanism for regulating the sensitivity of rods to light.     

Adeno-associated virus type 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression

Adeno-associated virus type 2 (AAV), a single-stranded, DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays an important role in AAV-mediated transgene expression (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997) and that a strong correlation exists between the phosphorylation state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et al., J. Virol. 72:1593-1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is mediated by the EGF-R PTK. Treatment of cells with EGF results in phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes dephosphorylation of the ssD-BP and consequently leads to increased expression of the transgene. Furthermore, AAV transduction efficiency inversely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of the ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These data suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vectors in human gene therapy.     

Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein

Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton-insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells.     

Synthesis of fluorescent substrates for protein tyrosine phosphatase assays

Two fluorescent substrates for protein tyrosine phosphatase (PTPase) reaction were prepared by conjugation of commercially available O-phosphotyrosine and dansyl chlorides. They were hydrolyzed by CD45 tyrosine phosphatase, and proved to be useful for PTPase assay.     

Shrinkage-induced protein tyrosine phosphorylation in Chinese hamster ovary cells

To investigate the signal transduction of osmotic stress, we examined hypertonicity-induced tyrosine phosphorylations in Chinese hamster ovary cells. Hyperosmosis elicited characteristic phosphotyrosine accumulation in at least 3 proteins (approximately 42, approximately 85, and approximately 120 kDa). The most prominent response occurred in the 85-kDa band (p85) whose phosphorylation was rapid, sustained, apparent already at mild hypertonicity (350 mosM), proportional to the extracellular osmotic concentration, and reversible. Hyperosmotic environment could not induce tyrosine phosphorylation if cell shrinkage was prevented by nystatin and appropriately composed media. Conversely, isotonic shrinkage caused strong tyrosine phosphorylation. Thus, the initial signal is a decrease in cell volume and not an increase in the intra- or extracellular osmotic concentration, or a rise in cytosolic K+ and Cl- levels. Tyrosine phosphorylation of p85 was not due to the hypertonicity-induced protein kinase C-dependent stimulation of the extracellular signal-regulated protein kinase, nor to the activation of stress-activated protein kinases. Tonicity-responsive proteins interacted with Grb2-glutathione S-transferase fusion proteins: the 120-kDa protein complexed with the SH2 and both SH3 domains, whereas p85 associated with the SH2 and the N-terminal SH3 domains of the adapter. Tyrosine phosphorylation of p85 is a sensitive indicator of reduced intracellular hydration and might signify a hitherto unrecognized, early volume-dependent signaling event.     

The second domain of the CD45 protein tyrosine phosphatase is critical for interleukin-2 secretion and substrate recruitment of TCR-zeta in vivo

The CD45 protein tyrosine phosphatase (PTPase) has been shown to regulate the activity of Lck and Fyn protein tyrosine kinases in T cells. However, it is not clear that these constitute the only CD45 substrates. Moreover, the manner by which PTPase activity and substrate recruitment are regulated, is poorly understood. Previous in vitro studies suggest that the first cytoplasmic PTPase domain (D1) of CD45 is the active PTPase, which may be regulated by an enzymatically inactive second PTPase domain (D2). However, the function of CD45 D2 in vivo is unknown. In this study, reconstitution of CD45(-) T cells with specific CD45 PTPase mutants allowed demonstration of a critical role for D2 in TCR-mediated interleukin (IL)-2 production. Specifically, replacement of CD45 D2 with that of the LAR PTPase to form a CD45/LAR:D2 chimera, abrogates CD45-dependent IL-2 production. This effect cannot be accounted for by loss of PTPase activity per se. The expression of D1 substrate-trapping mutants reveals an in vivo interaction between CD45 and TCR-zeta that is dependent on CD45 D2. Thus, cells expressing CD45 lacking D2 exhibit abnormal TCR-mediated signaling characterized by hyperphosphorylation of zeta and deficient ZAP-70 phosphorylation. These data suggest an essential role for CD45 D2 in TCR-regulated IL-2 production through substrate recruitment of the zeta chain.     

PSTPIP: a tyrosine phosphorylated cleavage furrow-associated protein that is a substrate for a PEST tyrosine phosphatase

We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system. This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow. The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase. PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP. This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase. Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow. Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. Finally, overexpression of mammalian PSTPIP in exponentially growing S. pombe results in a dominant-negative inhibition of cytokinesis. PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF.     

Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.     

Regulation of fibroblast motility by the protein tyrosine phosphatase PTP-PEST

The protein tyrosine phosphatase PTP-PEST is a cytosolic enzyme that displays a remarkable degree of selectivity for tyrosine-phosphorylated p130(Cas) as a substrate, both in vitro and in intact cells. We have investigated the physiological role of PTP-PEST using Rat1 fibroblast-derived stable cell lines that we have engineered to overexpress PTP-PEST. These cell lines exhibit normal levels of tyrosine phosphorylation of the majority of proteins but have significantly lower levels of tyrosine phosphorylation of p130(Cas) than control cells. Initial cellular events occurring following integrin-mediated attachment to fibronectin (cell attachment and spreading) are essentially unchanged in cells overexpressing PTP-PEST; similarly, the extent and time course of mitogen-activated protein kinase activation in response to integrin engagement is unchanged. In contrast, the reduced phosphorylation state of p130(Cas) is associated with a considerably reduced rate of cell migration and a failure of cells overexpressing PTP-PEST to accomplish the normally observed redistribution of p130(Cas) to the leading edge of migrating cells. Furthermore, cells overexpressing PTP-PEST demonstrate significantly reduced levels of association of p130(Cas) with the Crk adaptor protein. Our results suggest that one physiological role of PTP-PEST is to dephosphorylate p130(Cas), thereby controlling tyrosine phosphorylation-dependent signaling events downstream of p130(Cas) and regulating cell migration.     

Stimulation of NK-like YT cells via leukocyte function-associated antigen (LFA)-1. Possible involvement of LFA-1-associated tyrosine kinase in signal transduction after recognition of NK target cells

The YTA-1 anti-LFA-1 alpha mAb activates protein tyrosine kinase (PTK), augments NK cytotoxicity, and induces proliferation of fresh CD3- large granular lymphocytes. We demonstrate here that LFA-1 is physically associated in the YT human NK-like cell line cells with a PTK(s) that is distinct from Src family PTKs such as Lck, Fyn, or Lyn. In vitro kinase assays revealed similar association of protein kinase activity with LFA-1 in normal CD3- large granular lymphocytes. Tyrosine phosphorylation of the proteins associated with LFA-1 drastically increased in YT cells after stimulation with NK-sensitive K562 cells but not with NK-resistant P815 cells. Furthermore, pretreatment of YT cells with TS1/22 anti-LFA-1 alpha and TS1/18 anti-LFA-1 beta mAbs abrogated not only the cytotoxicity against K562 cells but also an increase in tyrosine phosphorylation of LFA-1-associated molecules induced by K562 stimulation. These results provide biochemical evidence that the PTK(s) associated with LFA-1 is involved in the signal transduction that follows the recognition of NK target cells.     

Replacement of tyrosine 1251 in the carboxyl terminus of the insulin-like growth factor-I receptor disrupts the actin cytoskeleton and inhibits proliferation and anchorage-independent growth

Insulin-like growth factor (IGF)-I signaling through the IGF-I receptor modulates cellular adhesion and proliferation and the transforming ability of cells overexpressing the IGF-I receptor. Tyrosine phosphorylation of intracellular proteins is essential for this transduction of the IGF-I-induced mitogenic and tumorigenic signals. IGF-I induces specific cytoskeletal structure and the phosphorylation of proteins in the associated focal adhesion complexes. The determination of the exact pathways emanating from the IGF-I receptor that are involved in mediating these signals will contribute greatly to the understanding of IGF-I action. We have previously shown that replacement of tyrosine residues 1250 and 1251 in the carboxyl terminus of the IGF-I receptor abrogates IGF-I-induced cellular proliferation and tumor formation in nude mice. In this study, replacement of either tyrosine 1250 or 1251 similarly reduces the cells ability to grow in an anchorage-independent manner. The actin cytoskeleton and cellular localization of vinculin are disrupted by replacement of tyrosine 1251. Tyrosine residues 1250 and 1251 are not essential for tyrosine phosphorylation of two known substrates; insulin receptor substrate-1 and SHC, nor association of known downstream adaptor proteins to these substrates. In addition, these mutant IGF-I receptors do not affect IGF-I-stimulated p42/p44 mitogen-activated protein kinase activation or phosphatidylinositol (PI) 3'-kinase activity. Thus, it appears that in fibroblasts expressing tyrosine 1250 and 1251 mutant IGF-I receptors, the signal transduction pathways impacting on mitogenesis and tumorigenesis do not occur exclusively through the PI 3'-kinase or mitogen-activated protein kinase pathways.     

p72syk protein tyrosine kinase: an early transducer of sIgG-triggered apoptotic signalling in human follicular lymphoma cells

Cross-linking of B cell antigen receptor (sIg) elicits different biological responses, including cell activation, proliferation, differentiation, anergy and cell death depending on the maturational stage of the cell. We established the tumor cell lines HF-1.3.4 and HF-4-9 from two patients with follicular lymphoma. Both cell lines carry the characteristic t(14;18) chromosomal translocation and display constitutively overexpressed Bcl-2. HF-1.3.4 represents a mature B cell with sIgG and several somatic hypermutations in its Ig genes, while HF-4-9 is a less mature B cell, expressing sIgM and only a few mutations in its Ig genes. Cross-linking of sIg with antibodies leads to apoptosis in HF-1.3.4 cells but not in HF-4-9 cells. Triggering of sIg induced, within seconds, identical tyrosine phosphorylation of p53/56lyn protein tyrosine kinase (PTK) and p55blk PTK in both of the cell lines; however, a prominent tyrosine phosphorylation and activation of p72syk PTK only in HF-1.3.4 cells. We conclude that p72syk PTK is of importance in relaying apoptotic signalling upon sIg cross-linking in the HF-1.3.4 cell line. Given the mature phenotype of the HF-1.3.4 cell line it serves as a model for the late negative selection during B cell ontogeny. Moreover, our results question the current concept that a constitutive overexpression of BcI-2 confers resistance to sIg ligation-induced apoptosis in lymphoma cells.     

Mitogenic response of murine B lymphocytes to Salmonella typhimurium lipopolysaccharide requires protein kinase C-dependent late tyrosine phosphorylations

Unlike the cross-linking of membrane immunoglobulins, the activation of B cells by lipopolysaccharide (LPS) does not involve the phosphoinositol turnover and the initial activation of tyrosine kinases. However, LPS-induced B-cell proliferation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A even when added 48 h after the beginning of the culture. Tyrosyl-phosphorylated proteins were detected by Western blotting after 24 h of culture with LPS, reaching a maximum concentration after 72 h. Late tyrosine phosphorylations were also detected in B cells activated for 72 h with anti-immunoglobulin M antibody and were abrogated by the protein synthesis inhibitor cycloheximide, the tyrosine kinase inhibitors genistein and herbimycin A, and the protein kinase C inhibitor chelerythrine. The role of protein kinase C in late tyrosine kinase activation is independent of Ca2+ mobilization and was confirmed by detection of a comparable but restricted pattern of tyrosine-phosphorylated substrates in B cells treated with phorbol myristate acetate alone or in association with ionomycin. Tyrosine kinase activation was dependent on de novo protein synthesis. However, culture supernatants of LPS-activated B cells were devoid of mitogenic activity and induced a phosphorylation pattern more restricted than that achieved by LPS. Altogether these data indicate that proliferation signals induced by LPS or by the cross-linking of membrane immunoglobulins are controlled by late tyrosine phosphorylations occurring throughout the first 3 days of culture, controlled in part by protein kinase C activation, and dependent on the synthesis of an intermediate protein(s) either not secreted in the culture supernatant or present but biologically inactive in naive B cells.