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PubMed1995

Interaction between G proteins and tyrosine kinases upon T cell receptor.CD3-mediated signaling.

Engagement of the T cell receptor (TCR).CD3 complex results in the induction of multiple intracellular events, with protein tyrosine kinases playing a pivotal role in their initiation. Biochemical studies also exist suggesting the involvement of heterotrimeric GTP-binding proteins (G proteins); however, the functional consequence of this participation in TCR.CD3-mediated signaling is unresolved. Here, we report TCR.CD3-mediated guanine nucleotide exchange among the 42-kDa G protein alpha subunits of the G alpha q/11 family, their physical association with CD3 epsilon, and the G alpha 11-dependent activation of phospholipase C beta. Protein tyrosine kinase inhibitors, however, abrogate TCR.CD3-mediated G protein activation. Quite interesting is the observation that cells transfected with a function-deficient mutant of G alpha 11 display diminished tyrosine phosphorylation of TCR.CD3 zeta and epsilon chains, as well as ZAP-70, upon anti-CD3 antibody triggering. These data indicate the involvement of the G alpha q/11 family in TCR.CD3 signaling at a step proximal to the receptor and suggest a reciprocal regulation between tyrosine kinases and G proteins in T cells.

Antibodies, Monoclonal↗
PubMed1996

The cytoplasmic tail of FcgammaRIIIAalpha is involved in signaling by the low affinity receptor for immunoglobulin G.

The low affinity receptor for IgG, FcgammaRIIIA, is a multimeric receptor composed of the ligand binding subunit FcgammaRIIIAalpha (CD16) in association with the signal-transducing subunits zeta or gamma. Previous studies suggested that the cytoplasmic tail of FcgammaRIIIAalpha was not required for FcgammaRIIIAalpha-zeta association or signaling by FcgammaRIIIA. However, in these studies, the truncated FcgammaRIIIAalpha chains still expressed the four most membrane-proximal amino acids of the cytoplasmic tail (amino acids 230-233). By successive truncations from the C terminus of FcgammaRIIIAalpha, we have studied the role played by the membrane-proximal amino acids of the cytoplasmic tail of FcgammaRIIIAalpha in (i) FcgammaRIIIA expression, (ii) FcgammaRIIIAalpha-zeta association, and (iii) signal transduction. We provide evidence that this region is not required for FcgammaRIIIA expression or FcgammaRIIIAalpha-zeta association. However, signaling by FcgammaRIIIA is strictly dependent on the membrane-proximal amino acids in the cytoplasmic tail of FcgammaRIIIAalpha. Thus, total deletion of the cytoplasmic tail of FcgammaRIIIAalpha results in a severely impaired tyrosine phosphorylation of phospholipase C-gamma1, zap, and syk and rise in intracellular free Ca2+ following receptor ligation with specific anti-CD16 monoclonal antibody or Ig-anti-Ig complexes, suggesting that FcgammaRIIIAalpha-zeta association per se is not sufficient to establish the signal function of FcgammaRIIIA. In conclusion, the present findings demonstrate that the most membrane-proximal amino acids of the FcgammaRIIIAalpha cytoplasmic tail play a critical role in ligand-induced signal transduction by the FcgammaRIIIAalpha-zeta complex.

Amino Acid Sequence↗
PubMed1996

Identification of the site in the Syk protein tyrosine kinase that binds the SH2 domain of Lck.

The Syk protein tyrosine kinase (PTK) is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. Upon cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues and enzymatically activated. We and others have found that the Lck kinase, a member of the Src family of PTKs, binds through its Src homology (SH) 2 domain to tyrosine phosphorylated Syk and to the related Zap kinase. Here we report that this interaction is direct and identify the two tandem tyrosines at the autophosphorylation site of Syk, Tyr518, and Tyr519, as the binding site for the SH2 domain of Lck. Mutation of either or both tyrosines to phenylalanines abrogated binding, while mutation of a second repetition of the motif at Tyr539 and Tyr540, or of the three tyrosines in the C terminus of Syk, did not. The SH2 domain of Lck bound the autophosphorylation site only when both Tyr518 and Tyr519 were phosphorylated. In intact cells the binding of the SH2 domain of Lck correlated with the ability of Syk to induce tyrosine phosphorylation of cellular proteins.

Amino Acid Sequence↗
PubMed1996

Signaling through CD44 is mediated by tyrosine kinases. Association with p56lck in T lymphocytes.

Evidence from a large body of studies indicates that CD44 is involved in a number of important biological processes, including lymphocyte activation and homing, hematopoiesis, and tumor progression and metastasis. A proper understanding of the role of CD44 in these processes has been severely hampered by a lack of insight into the mode in which CD44 communicates with intracellular signal transduction pathways. In this report, we have addressed this aspect of CD44 functioning by studying CD44 signaling in T lymphocytes. We show that ligation of CD44 by monoclonal antibodies (mAbs) transduces signals to T cells which lead to tyrosine phosphorylation of ZAP-70 and other intracellular proteins. In vitro kinase assays demonstrate that cross-linking of CD44 induces an increase in the intrinsic activity of p56lck. Furthermore, immunoprecipitations show that CD44 is physically associated with p56lck. Our findings suggest that tyrosine kinases, particularly p56lck, play a central role in CD44 mediated signaling.

Animals↗
PubMed1996

Syk, activated by cross-linking the B-cell antigen receptor, localizes to the cytosol where it interacts with and phosphorylates alpha-tubulin on tyrosine.

Syk (p72syk) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate-treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as alpha-tubulin. alpha-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. alpha-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including alpha-tubulin.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and expression of mouse and human cDNAs encoding heparan sulfate D-glucosaminyl 3-O-sulfotransferase.

The cellular rate of anticoagulant heparan sulfate proteoglycan (HSPGact) generation is determined by the level of a kinetically limiting microsomal activity, HSact conversion activity, which is predominantly composed of the long sought heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063-27071; Liu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27072-27082). Mouse 3-OST cDNAs were isolated by proteolyzing the purified enzyme with Lys-C, sequencing the resultant peptides as well as the existing amino terminus, employing degenerate polymerase chain reaction primers corresponding to the sequences of the peptides as well as the amino terminus to amplify a fragment from LTA cDNA, and utilizing the resultant probe to obtain full-length enzyme cDNAs from a lambda Zap Express LTA cDNA library. Human 3-OST cDNAs were isolated by searching the expressed sequence tag data bank with the mouse sequence, identifying a partial-length human cDNA and utilizing the clone as a probe to isolate a full-length enzyme cDNA from a lambda TriplEx human brain cDNA library. The expression of wild-type mouse 3-OST as well as protein A-tagged mouse enzyme by transient transfection of COS-7 cells and the expression of both wild-type mouse and human 3-OST by in vitro transcription/translation demonstrate that the two cDNAs directly encode both HSact conversion and 3-OST activities. The mouse 3-OST cDNAs exhibit three different size classes because of a 5'-untranslated region of variable length, which results from the insertion of 0-1629 base pairs (bp) between residues 216 and 217; however, all cDNAs contain the same open reading frame of 933 bp. The length of the 3'-untranslated region ranges from 301 to 430 bp. The nucleic acid sequence of mouse and human 3-OST cDNAs are approximately 85% similar, encoding novel 311- and 307-amino acid proteins of 35,876 and 35,750 daltons, respectively, that are 93% similar. The encoded enzymes are predicted to be intraluminal Golgi residents, presumably interacting via their C-terminal regions with an integral membrane protein. Both 3-OST species exhibit five potential N-glycosylation sites, which account for the apparent discrepancy between the molecular masses of the encoded enzyme (approximately 34 kDa) and the previously purified enzyme (approximately 46 kDa). The two 3-OST species also exhibit approximately 50% similarity with all previously identified forms of the heparan biosynthetic enzyme N-deacetylase/N-sulfotransferase, which suggests that heparan biosynthetic enzymes share a common sulfotransferase domain.

Amino Acid Sequence↗
PubMed1997

Regulation of the low molecular weight phosphotyrosine phosphatase by phosphorylation at tyrosines 131 and 132.

Activation of resting T lymphocytes is initiated by rapid but transient tyrosine phosphorylation of a number of cellular proteins. Several protein tyrosine kinases and protein tyrosine phosphatases are known to be important for this response. Here we report that normal T lymphocytes express the B isoform of low molecular weight protein tyrosine phosphatase B (LMPTP-B). The cDNA was cloned from Jurkat T cells, and an antiserum was raised against it. LMPTP immunoprecipitated from resting Jurkat T cells was found to be tyrosine phosphorylated. On stimulation of the cells through their T cell antigen receptor, the phosphotyrosine content of LMPTP-B declined rapidly. In co-transfected COS cells, Lck and Fyn caused phosphorylation of LMPTP, whereas Csk, Zap, and Jak2 did not. Most of the phosphate was located at Tyr-131, and some was also located at Tyr-132. Incubation of wild-type LMPTP with Lck and adenosine 5'-O-(thiotriphosphate) caused a 2-fold increase in the activity of LMPTP. Site-directed mutagenesis showed that Tyr-131 is important for the catalytic activity of LMPTP, and that thiophosphorylation of Tyr-131, and to a lesser degree Tyr-132, is responsible for the activation.

3T3 Cells↗
PubMed1998

Fyn, Yes, and Syk phosphorylation sites in c-Cbl map to the same tyrosine residues that become phosphorylated in activated T cells.

Protooncogenic protein c-Cbl undergoes tyrosine phosphorylation in response to stimulation through the receptors for antigens, immunoglobulins, cytokines, and growth factors as well as through the integrins. Tyrosine phosphorylation of c-Cbl may play a functional role in signal transduction, since c-Cbl interacts with many crucial signaling molecules including protein-tyrosine kinases, adaptor proteins, and phosphatidylinositol 3'-kinase. Therefore, it is essential for our understanding of the functions of c-Cbl in signal transduction to identify its tyrosine phosphorylation sites, to determine the protein-tyrosine kinases that phosphorylate these sites, and to elucidate the role of these sites in the interactions of c-Cbl with other signaling proteins. In this report, we demonstrate that tyrosines 700, 731, and 774 are the major tyrosine phosphorylation sites of c-Cbl in T cells in response to pervanadate treatment, as well as in response to TcR/CD3 ligation. Coexpression experiments in COS cells demonstrate that among T cell-expressed Src- and Syk-related protein-tyrosine kinases, Fyn, Yes, and Syk appear to play a major role in phosphorylation of c-Cbl, whereas Lck and Zap phosphorylate c-Cbl ineffectively. Fyn, Yes, and Syk phosphorylate the same sites of c-Cbl that become phosphorylated in stimulated T cells. Among these kinases, Fyn and Yes demonstrate strong binding to c-Cbl, which involves both phosphotyrosine-dependent and phosphotyrosine-independent mechanisms.

Amino Acid Sequence↗
PubMed1998

Coordinated regulation of the tyrosine phosphorylation of Cbl by Fyn and Syk tyrosine kinases.

Cross-linking of the T cell antigen receptor (TCR)-CD3 complex induces rapid tyrosine phosphorylation and activation of Src (Lck and Fyn) and Syk (Syk and Zap-70) family protein tyrosine kinases (PTKs) which, in turn, phosphorylate multiple intracellular substrates. Cbl is a prominent PTK substrate suggesting a pivotal role for it in early signal transduction events. However, the regulation of Cbl function and tyrosine phosphorylation in T cells by upstream PTKs remains poorly understood. In the present study, we used genetic and biochemical approaches to demonstrate that Cbl directly interacts with Syk and Fyn via its N-terminal and C-terminal regions, respectively. Tyr-316 of Syk was required for the interaction with Cbl as well as for the maximal tyrosine phosphorylation of Cbl. However, both wild-type Syk and Y316F-mutated Syk phosphorylated equally well the C-terminal fragment of Cbl in vivo, suggesting the existence of an alternative, N terminus-independent mechanism for the Syk-induced tyrosine phosphorylation of Cbl. This mechanism appears to involve Fyn, since, in addition to its association with the C-terminal region of Cbl, Fyn also associated with Syk and enhanced the Syk-induced tyrosine phosphorylation of Cbl. These findings implicate Fyn as an adaptor protein that facilitates the interaction between Syk and Cbl, and suggest that Src and Syk family PTKs coordinately regulate the tyrosine phosphorylation of Cbl.

Cloning, Molecular↗
PubMed1998

Tandem SH2 domains confer high specificity in tyrosine kinase signaling.

SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase-linked receptors. Recent three-dimensional structures suggest mechanisms by which tandem SH2 domains might confer higher specificity than individual SH2 domains. To test this, binding studies were conducted with tandem domains from the five signaling enzymes: phosphatidylinositol 3-kinase p85, ZAP-70, Syk, SHP-2, and phospholipase C-gamma1. Bisphosphorylated TAMs (tyrosine-based activation motifs) were derived from biologically relevant sites in platelet-derived growth factor, T cell, B cell, and high affinity IgE receptors and the receptor substrates IRS-1 (insulin receptor substrate-1) and SHPS-1/SIRP. Each tandem SH2 domain binds a distinct TAM corresponding to its appropriate biological partner with highest affinity (0.5-3.0 nM). Alternative TAMs bind the tandem SH2 domains with 1,000- to >10,000-fold lower affinity than biologically relevant TAMs. This level of specificity is significantly greater than the approximately 20-50-fold typically seen for individual SH2 domains. We conclude that high biological specificity is conferred by the simultaneous interaction of two SH2 domains in a signaling enzyme with bisphosphorylated TAMs in activated receptors and substrates.

Amino Acid Sequence↗
PubMed1998

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.

Humans↗
PubMed1999

FYN-T-FYB-SLP-76 interactions define a T-cell receptor zeta/CD3-mediated tyrosine phosphorylation pathway that up-regulates interleukin 2 transcription in T-cells.

Protein-tyrosine kinases p56(Lck), SYK, and ZAP-70 and downstream adaptors LAT and SLP-76 have been implicated as essential components in T-cell activation. Another lymphoid-specific adaptor FYB/SLAP has also been identified as a predominant binding partner of SLP-76 and the Src kinase FYN-T, although its role in the activation process has been unclear. In this study, we demonstrate that FYN-T selectively phosphorylates FYB providing a template for the recruitment of FYN-T and SLP-76 SH2 domain binding. This interaction is unusual in its distinct cytoplasmic localization and its long term stable kinetics of phosphorylation. Furthermore, we demonstrate for the first time that the co-expression of all three components of the FYN-T-FYB-SLP-76 matrix can synergistically up-regulate T-cell receptor-driven interleukin 2 transcription activity. These findings document the existence of a T-cell receptor-regulated FYN-T-FYB pathway that interfaces with the adaptor SLP-76 and up-regulates lymphokine production in T-cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Cloning of linoleate diol synthase reveals homology with prostaglandin H synthases.

Linoleate diol synthase is a homotetrameric ferric hemeprotein, which catalyzes dioxygenation of linoleic acid to (8R)-hydroperoxylinoleate and isomerization of the hydroperoxide to (7S,8S)-dihydroxylinoleate. Ferryl intermediates and a tyrosyl radical are formed in the reaction. Linoleate diol synthase was digested with endoproteinase Lys-C, and internal peptides were sequenced. The sequence information was used for reverse transcription-polymerase chain reaction analysis, and a cDNA probe was obtained. Northern blot analysis of linoleate diol synthase suggested a 3.7-kilobase pair (kb) mRNA. A full-length clone of the linoleate diol synthase gene was obtained by screening of a genomic lambda-ZAP II library of the fungus Gaeumannomyces graminis. The 5'-untranslated region contained CAAT- and TATA-like boxes. The gene contained three short introns and spanned over 3.2-kb. The deduced open reading frame consisted of 2.9-kb, which corresponded to 978 amino acids and a molecular subunit mass of 108,000. Data base analysis with the gapped BLAST algorithm showed that 391 residues of linoleate diol synthase was 23-24% identical and 36-37% positive with the catalytic domain of mammalian prostaglandin H (PGH) synthase-2. Based on homology with PGH synthases, the proximal heme ligand of linoleate diol synthase was tentatively identified as His-379 and the important tyrosine for catalysis as residue 376 (apparent consensus EFNXXXYXWH). The distal heme ligand was tentatively identified as His-203 (apparent consensus THXXFXT). We conclude from catalytic and structural similarities that linoleate diol synthase and PGH synthases likely share common ancestry and may belong to a gene family of fatty acid heme dioxygenases.

Amino Acid Sequence↗
PubMed1999

Novel isoform of lymphoid adaptor FYN-T-binding protein (FYB-130) interacts with SLP-76 and up-regulates interleukin 2 production.

T-cell activation involves the participation of protein-tyrosine kinases p56(lck) and ZAP-70/SYK as well as lymphoid proteins such as SLP-76 and FYB/SLAP. FYB/SLAP has the hallmarks of an adaptor protein that binds to the SH2 domains of the Src kinase FYN-T and SLP-76. Whereas two forms of FYB at 120 and 130 kDa have been identified biochemically, a cDNA encoding only the lower molecular weight isoform has been cloned (termed FYB-120 or SLAP-130). In this study, we report the isolation of an alternative isoform of FYB with a molecular mass of 130 kDa (FYB-130) that has the same structure as FYB-120 except for an insertion of 46 amino acids toward the carboxyl-terminal region of the protein. FYB-120 and FYB-130 share an ability to bind to the SH2 domains of FYN-T and SLP-76, to act as substrates for p59(FYN-T), and to be expressed in the cytoplasm and nucleus of T-cells. Differences were noted between the isoforms in the efficiency of binding to SLP-76 and in the preferential expression of FYB-130 in mature T-cells. When co-expressed together with FYN-T and SLP-76, FYB-130 caused a significant increase in anti-CD3-driven NF-AT transcription. Finally, fluorescence in situ hybridization analysis localized the FYB gene to human chromosome 5 at position p13.1. FYB-130 therefore represents a novel variant of FYB protein that can up-regulate T-cell receptor-driven interleukin 2 production in mature T-cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Requirement of a Src family kinase for initiating calcium release at fertilization in starfish eggs.

Signal transduction leading to calcium release in echinoderm eggs at fertilization requires phospholipase Cgamma-mediated production of inositol trisphosphate (IP(3)), indicating that a tyrosine kinase is a likely upstream regulator. Because previous work has shown a fertilization-dependent association between the Src homology 2 (SH2) domains of phospholipase Cgamma and a Src family kinase, we examined whether a Src family kinase was required for Ca(2+) release at fertilization. To inhibit the function of kinases in this family, we injected starfish eggs with the SH2 domains of Src and Fyn kinases. This inhibited Ca(2+) release in response to fertilization but not in response to injection of IP(3). We further established the specificity of the inhibition by showing that the SH2 domains of several other tyrosine kinases (Abl, Syk, and ZAP-70), and the SH3 domain of Src, were not inhibitory. Also, a point-mutated Src SH2 domain, which has reduced affinity for phosphotyrosine, was a correspondingly less effective inhibitor of fertilization-induced Ca(2+) release. These results indicate that a Src family kinase, by way of its SH2 domain, links sperm-egg interaction to IP(3)-mediated Ca(2+) release at fertilization in starfish eggs.

Animals↗
PubMed2000

Evidence that Llck-mediated phosphorylation of p56dok and p62dok may play a role in CD2 signaling.

The Lck tyrosine kinase is involved in signaling by T cell surface receptors such as TCR/CD3, CD2, and CD28. As other downstream protein-tyrosine kinases are activated upon stimulation of these receptors, it is difficult to assign which tyrosine-phosphorylated proteins represent bona fide Lck substrates and which are phosphorylated by other tyrosine kinases. We have developed a system in which Lck can be activated independently of TCR/CD3. We have shown that activation of an epidermal growth factor receptor/Lck chimera leads to the specific phosphorylation of Ras GTPase-activating protein (RasGAP) and two RasGAP-associated proteins, p56(dok) and p62(dok). Activation of the chimeric protein correlates with an increase in cellular Ca(2+) in the absence of ZAP-70 and phospholipase Cgamma1 phosphorylation. Furthermore, we have found that p62(dok) co-immunoprecipitates with the activated epidermal growth factor receptor/LckF505 and that phosphorylated Dok proteins bind to the Src homology 2 domain of Lck in vitro. In addition, we have shown that activation via the CD2 but not the TCR/CD3 receptor leads to the phosphorylation of p56(dok) and p62(dok). Using JCaM1.6 cells, we have demonstrated that Lck is required for CD2-mediated phosphorylation of Dok proteins. We propose that phosphorylation and Src homology 2-mediated association of p56(dok) and p62(dok) with Lck play a selective function in accessory receptor signal transduction mechanisms.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Regulation of T cell receptor- and CD28-induced tyrosine phosphorylation of the focal adhesion tyrosine kinases Pyk2 and Fak by protein kinase C. A role for protein tyrosine phosphatases.

The T cell receptor (TCR)-CD3 complex and the costimulatory molecule CD28 are critical for T cell function. Both receptors utilize protein tyrosine kinases (PTKs) for the phosphorylation of various signaling molecules, a process that is critical for the function of both receptors. The PTKs of the focal adhesion family, Pyk2 and Fak, have been implicated in the signaling of TCR and CD28. We show here evidence for the regulation of TCR- and CD28-induced tyrosine phosphorylation of the focal adhesion PTKs by protein kinase C (PKC). Thus, treating Jurkat T cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) rapidly and strongly reversed receptor-induced tyrosine phosphorylation of the focal adhesion PTKs. In contrast, PMA did not affect TCR-induced tyrosine phosphorylation of CD3zeta or the PTKs Fyn and Zap-70. However, PMA induced a strong and rapid dephosphorylation of the linker molecule for activation of T cells. PMA failed to induce the dephosphorylation of proteins in PKC-depleted cells or in cells pretreated with the PKC inhibitor Ro-31-8220, confirming the role of PKC in mediating the PMA effect on receptor-induced protein tyrosine phosphorylation. The involvement of protein tyrosine phosphatases (PTPases) in mediating the dephosphorylation of the focal adhesion PTKs was confirmed by the failure of PMA to dephosphorylate Pyk2 in cells pretreated with the PTPase inhibitor orthovanadate. These results implicate PKC in the regulation of receptor-induced tyrosine phosphorylation of the focal adhesion PTKs in T cells. The data also suggest a role for PTPases in the PKC action.

Antigens, CD↗
PubMed2000

The major chicken egg envelope protein ZP1 is different from ZPB and is synthesized in the liver.

The extracellular matrix surrounding vertebrate oocytes is called the zona pellucida in mammals and perivitelline membrane (pvm) in birds. We have analyzed this structure in chicken follicles and laid eggs and have identified a 95-kDa component of the pvm, which, by protein sequencing, shows homology to mammalian zona pellucida proteins. Surprisingly, we could not detect this protein in ovarian granulosa cells or oocytes but instead found high levels in the liver of the laying hen. In contrast, it is absent in rooster liver but can be efficiently induced by estrogen treatment of the animal. An immunoscreen of a liver lambda-ZAP library yielded a cDNA coding for a protein of 934 amino acids. It displayed significant homology to members of the ZP1/ZPB family from other species, notably to mouse and rat ZP1, and was therefore designated chkZP1. It is clearly different from a protein designated chkZPB that had been deposited in the data base previously. Alignment of the known members of the ZP1/ZPB family demonstrated the existence of at least three subgroups, with representatives of both the ZP1 and the ZPB sequence homology group occurring in vertebrates. Northern blot analysis of liver extracts revealed the presence of a single 3. 2-kilobase mRNA coding for chkZP1, distinct from the chkZPB transcript detectable in follicles. Immunohistochemical analysis of follicle sections demonstrates that chkZP1 can be found in the blood vessels of the theca cell layer as well as in the pvm surrounding the oocyte. Thus, in the chicken, at least one of the major pvm components is synthesized in the liver and is transported via the bloodstream to the follicle.

Amino Acid Sequence↗