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PubMed2004

High-throughput analysis of genome-wide receptor tyrosine kinase expression in human cancers identifies potential novel drug targets.

Novel high-throughput analyses in molecular biology allow sensitive and rapid identification of disease-related genes and drug targets. We have used quantitative real-time reverse transcription-PCR reactions (n = 23000) to analyze expression of all human receptor tyrosine kinases (n = 56) in malignant tumors (n = 313) of different origins and normal control samples (n = 58). The different tumor types expressed very different numbers of receptor tyrosine kinases: whereas brain tumors and testicular cancer expressed 50 receptor tyrosine kinases, acute myeloid leukemia (AML) samples expressed only 20 different ones. Specimens of similar tumor origin exhibited characteristic receptor tyrosine kinase expression patterns and were grouped together in hierarchical cluster analyses. When we focused on specific tumor entities, receptor tyrosine kinases were identified that were disease and/or stage specific. Leukemic blasts from AML bone marrow samples differed significantly in receptor tyrosine kinase expression compared with normal bone marrow and purified CD34+ cells. Among the differentially expressed receptor tyrosine kinases, we found FLT3, c-kit, CSF1 receptor, EPHB6, leukocyte tyrosine kinase, and ptk7 to be highly overexpressed in AML samples. Whereas expression changes of some of these were associated with altered differentiation patterns (e.g., CSF1 receptor), others, such as FLT3, were genuinely overexpressed in leukemic blasts. These data and the associated database (http://medweb.uni-muenster.de/institute/meda/research/) provide a comprehensive view of receptor tyrosine kinase expression in human cancer. This information can assist in the definition of novel drug targets.

Antigens, CD34↗
PubMed1995

Evidence that tyrosine phosphorylation may increase tight junction permeability.

Tight junction permeability control is important in a variety of physiological and pathological processes. We have investigated the role of tyrosine phosphorylation in the regulation of tight junction permeability. MDCK epithelial cells and brain endothelial cells were grown on filters and tight junction permeability was determined by transcellular electrical resistance (TER). The tyrosine phosphatase inhibitor pervanadate caused a concentration- and time-dependent decrease in TER in both MDCK and brain endothelial cells. However, as expected, pervanadate resulted in the tyrosine phosphorylation of many proteins; hence interpretation of its effects are extremely difficult. Phenylarsine oxide, a more selective tyrosine phosphatase inhibitor, caused the tyrosine phosphorylation of relatively few proteins as analyzed by immunoblotting of whole cell lysates. This inhibitor, like pervanadate, also elicited a decrease in TER in the two cell types. In the MDCK cells, the action of phenylarsine oxide could be reversed by the subsequent addition of the reducing agent 2,3-dimercaptopropanol. Immunocytochemistry revealed that phenylarsine oxide rapidly stimulated the tyrosine phosphorylation of proteins associated with intercellular junctions. Because of the known influence of the adherens junction on tight junctions, we analyzed immunoprecipitates of the E-cadherin/catenin complex from MDCK cells treated with phenylarsine oxide. This revealed an increase in the tyrosine phosphorylation of beta-catenin, but not of alpha-catenin. However, the tight junction associated protein ZO-1 was also tyrosine phosphorylated after PAO treatment. These data indicate that tight junction permeability may be regulated via mechanisms involving tyrosine phosphorylation of adherens junction and tight junction proteins.

Animals↗
PubMed2004

A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).

The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a 'T'-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the 'T'-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.

Animals↗
PubMed1999

Differential substrate recognition capabilities of Janus family protein tyrosine kinases within the interleukin 2 receptor (IL2R) system: Jak3 as a potential molecular target for treatment of leukemias with a hyperactive Jak-Stat signaling machinery.

Substrate recognition by Janus family protein tyrosine kinases was examined utilizing recombinant baculovirus produced components of the interleukin 2 receptor (IL2R) system i.e. Jak1, Transducers and Activators of Transcription (STAT). Wild type Jak3 was able to tyrosine phosphorylate a kinase-dead Jak1 (Jak1E908). In contrast wild type Jak1 was unable to tyrosine phosphorylate kinase dead Jak3 (Jak3E851). This unilateral transphosphorylation between Jak3 and Jak1 prompts the hypothesis that in the IL2R system the activation of Jak3 precedes Jak1 activation. Both the IL2Rbeta and IL2Rgammac subunits underwent tyrosine phosphorylation when co-expressed with wild-type Jak3. By comparison only IL2Rbeta was recognized and tyrosine phosphorylated by wild-type Jak1. These results are consistent with the notion that Jakl is pre-associated with IL2Rbeta and Jak3 is pre-associated with IL2Rgammac. STAT1, STAT3, and STAT5 underwent tyrosine phosphorylation when co-expressed with Jakl and therefore are substrates for the respective Jak kinases. In contrast, Jak3 co-expression resulted in tyrosine phosphorylation of STAT3 and STAT5 but not STAT1. Notably a polypeptide representing the kinase domain of Jak3 (Jak3-JH1) gained the ability to tyrosine phosphorylate STAT1, suggesting that the changes in substrate recognition may be influenced by domains outside the kinase domain. These findings provide evidence that Jak1 and Jak3 differentially recognize specific substrates, thereby having the ability to contribute specific signals, and the substrate specificity may be influenced by multiple domains of these tyrosine kinases.

Amino Acid Sequence↗
PubMed2002

Proline-rich tyrosine kinase 2 and focal adhesion kinase are involved in different phases of platelet activation by vWF.

Stimulation of human platelets with von Willebrand factor (vWF) induces the rapid tyrosine phosphorylation of several proteins, but very little is known on the tyrosine kinases involved in this process. In the present work, we investigated and compared the activation of two related tyrosine kinases expressed in platelets: the proline-rich tyrosine kinase 2 (Pyk2) and the focal adhesion kinase (FAK). Both kinases were tyrosine phosphorylated upon vWF interaction with glycoprotein Ib-IX-V complex, but with different mechanisms. Tyrosine phosphorylation of FAK was totally dependent on thromboxane A2 production, and was inhibited by the integrin alphaIIbeta3 antagonist RGDS peptide. Moreover, chelation of intracellular calcium or inhibition of protein kinase C (PKC) totally blocked vWF-induced tyrosine phosphorylation of FAK, indicating that this event is downstream phospholipase A2 and phospholipase C activation. By contrast, tyrosine phosphorylation of Pyk2 was only partially reduced by aspirin and RGDS, and was not affected by either calcium chelation or PKC inhibition, suggesting that activation of this kinase does not require phospholipase-mediated signalling. Both FAK and Pyk2 translocated to the cytoskeleton upon vWF stimulation of human platelets by a mechanism depending on agonist-induced actin polymerisation. Prevention of cytoskeletal relocation of Pyk2 and FAK by cytochalasin D totally blocked vWF-induced tyrosine phosphorylation of both kinases. Finally, phosphorylation of Pyk2 induced by vWF, but not by thrombin, was inhibited by piceatannol, suggesting that this kinase lies downstream Syk. These results demonstrate that both Pyk2 and FAK are involved in platelet stimulation by vWF, but indicate that only Pyk2 may play a role in the early signal transduction events activated by ligand binding to glycoprotein Ib-IX-V.

Blood Platelets↗
PubMed1992

Antigen receptor-mediated protein tyrosine kinase activity is regulated by a pertussis toxin-sensitive G protein.

Ligation of the Ag receptor on B cells is associated with a rapid increase in phosphorylation on tyrosine residues of multiple substrates. One of the substrates is the phosphoinositide-specific phospholipase C-gamma 1. Because activation of phospholipase C-gamma 1 seems to be dependent on tyrosine phosphorylation, it is assumed that the two signaling pathways, phosphatidylinositol turnover and tyrosine phosphorylation, might be linked. However, since the Ag receptor does not possess a kinase domain, it remains unclear how these signaling pathways are regulated by the Ag receptor. Previous studies have proposed the existence of a receptor-coupled G protein that regulates inositol phosphate production in B cells. We confirm that phosphoinositide turnover is regulated by a pertussis toxin (PT)-sensitive G protein, most probably by controlling phosphorylation of phospholipase C-gamma 1. We show that treatment of permeabilized B cells with a nonhydrolyzable GTP analogue guanosine 5'-[3-thio]triphosphate induced an increase in tyrosine phosphorylation of multiple substrates that are identical to the proteins phosphorylated after anti-IgM stimulation. Furthermore, binding of the inactive form of G proteins with guanosine 5'-[2-thio]-triphosphate blocked anti-IgM induced tyrosine phosphorylation in permeabilized B cells. The results indicate that an Ag receptor-coupled G protein controls protein tyrosine kinase activity. We show that this G protein is sensitive to PT because tyrosine phosphorylation mediated by the Ag receptor was inhibited by this toxin in a concentration-dependent manner. Similar concentrations of PT also blocked tyrosine phosphorylation on phospholipase C-gamma 1 and generation of inositol phosphates. Preincubation of intact B cells with PT resulted in inhibition of c-fos mRNA expression and DNA synthesis in anti-IgM stimulated B cells, indicating that post-transcriptional events are also controlled by the Ag-receptor coupled G protein. We conclude that Ag receptor-associated protein tyrosine kinase activity is regulated by a G protein. This PT-sensitive G protein also regulates phosphorylation and activation of phospholipase C-gamma 1 as well as later events in B cell activation such as c-fos mRNA expression and proliferation.

B-Lymphocytes↗
PubMed1992

Interferon alpha induces rapid tyrosine phosphorylation of the alpha subunit of its receptor.

The mechanisms of generation of second messengers after binding of interferon alpha (IFN alpha) to its receptor remain unknown. We have studied the phosphorylation of the alpha subunit of the IFN alpha receptor, which is recognized by the monoclonal antibody IFNa receptor 3. Immunoblotting experiments showed that IFN alpha induced rapid tyrosine phosphorylation of the alpha subunit in the IFN alpha-sensitive H-929, U-266, and Daudi cell lines. Immunoprecipitation experiments performed with 32P-labeled cells showed that the alpha subunit is phosphorylated before IFN alpha treatment and that the level of phosphorylation increases after IFN alpha stimulation. Phosphoamino acid analysis confirmed the IFN alpha-induced tyrosine phosphorylation and demonstrated that the base-line phosphorylation corresponded to serine phosphorylation that increased 50% upon IFN alpha treatment. Tyrosine phosphorylation of the alpha subunit was time- and dose-dependent, further demonstrating the specificity of the process. Phosphorylation of the alpha subunit of the receptor occurred rapidly after IFN alpha binding, both at 37 and 4 degrees C. Exposure of the cells to the tyrosine kinase inhibitor genistein blocked the IFN alpha-induced tyrosine phosphorylation of this subunit of the IFN alpha receptor. In contrast H7, a specific protein kinase C inhibitor, and acute and chronic exposure to phorbol esters had no effect on tyrosine phosphorylation, suggesting that protein kinase C does not regulate the tyrosine phosphorylation of the alpha subunit of the IFN alpha receptor. No IFN alpha-induced tyrosine phosphorylation was observed in the IFN alpha-resistant U-937 cell line that expresses a variant IFN alpha receptor. Altogether these data suggest that tyrosine phosphorylation of the alpha subunit may play a role in the signal transduction pathway of IFN alpha.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Myeloid differentiation associated tyrosine protein kinase activity in WEHI-3B murine monomyelocytic leukemia cells.

Tyrosine protein kinase activity was examined during the induction of granulocytic differentiation of WEHI-3B murine monomyelocytic leukemia cells by retinoic acid and aclacinomycin A. Tyrosine kinase activity was found to increase throughout the period of induced maturation. The specificity of this increase in enzymatic activity for the differentiated state was demonstrated by the findings that (a) it was independent of the inducer used, and (b) the treatment of a differentiation-resistant subline of this murine leukemia with an inducer did not produce a significant elevation of tyrosine kinase activity. To determine whether tyrosine protein kinase activity was involved in the differentiation process itself or whether it was a product of the mature state, a series of experimental approaches was employed. Kinetic analyses showed that tyrosine protein kinase activity continued to increase beyond the peak in the level of differentiation. In addition, the total cellular protein phosphotyrosine content, measured by immunoblotting and flow cytometric analysis with anti-phosphotyrosine antibodies, did not increase in accord with the elevation of tyrosine kinase activity. Increases in protein phosphotyrosine content, which were dependent upon the length of exposure to the inducing agent, were observed when cultured cells were treated with the phosphotyrosine phosphatase inhibitor, sodium orthovanadate. Thus, the effect of the increasing tyrosine kinase activity in maturing cells appeared to be negated by competing protein phosphotyrosine phosphatase activity. Finally, the inhibitors of tyrosine kinase activity, genistein and PKI-23, did not interfere with the induction of differentiation by retinoic acid. These findings support the concept that myeloid differentiation associated tyrosine protein kinase activity may not be involved in the initiation of the differentiation process itself. This conclusion deviates from previous assumptions, based on earlier work in this laboratory as well as in that of others, that the differentiation associated kinase activity has an essential role in the initiation of the maturation process. An attractive alternative speculation is that this activity may have a functional role in the mature myeloid cell.

Animals↗
PubMed1987

Tyrosine kinase and phosphotyrosine phosphatase activity in human promyelocytic leukemia cells and human polymorphonuclear leukocytes.

Although an increase in protein phosphorylation on tyrosine was first noted as a result of cell transformation or the application of growth factors to cells, recent reports have shown high levels of tyrosine kinases in nondividing tissues. For that reason, we have investigated whether normal human polymorphonuclear leukocytes (PMN) contain tyrosine kinase and phosphatase activity. Using a copolymer of glutamine: tyrosine as a substrate for the phosphotransferase reaction, we have demonstrated that PMN contain a cytosolic tyrosine kinase activity that elutes as a single peak from Sephacryl S-200 chromatography and has a molecular weight of 70 kilodaltons. Human promyelocytic leukemia cells (HL-60), contain a similar activity (as demonstrated by column chromatography), with only 25% of the activity found in PMN. This cytosolic tyrosine kinase can phosphorylate angiotensin II and a fragment of the src protein containing tyrosine 416, which suggests a similar substrate specificity to other tyrosine-phosphorylating protein kinases. In addition, we have demonstrated that PMN have double the amount of phosphotyrosine phosphatase (PTPase) activity of that found in HL-60 cells. This enzyme has a Km of 0.932 mmol/L and a Vmax of 0.355 mumol inorganic phosphate released/mg protein/min, which is similar to other cellular PTPase. Activation of PMN with f-Met-Leu-Phe and phorbol esters causes a slight but statistically significant drop in PMN PTPase activity. These results suggest that terminally differentiated myeloid cells have high tyrosine kinase and phosphatase activity, which may play a role in stimulus response coupling in the mature PMN.

Cell Line↗
PubMed1993

Inhibition of bradykinin- and thapsigargin-induced Ca2+ entry by tyrosine kinase inhibitors.

We examined the involvement of tyrosine kinase activity in the bradykinin (BK)-mediated signal transduction process. Immunoblots with anti-phosphotyrosine antibodies following BK stimulation of human fibroblasts showed tyrosine phosphorylation of specific proteins that could be inhibited by the tyrosine kinase inhibitors genistein and tyrphostin. Image analysis data from individual cells stimulated by BK in the presence of genistein and tyrphostin showed that these inhibitors block the plateau phase but not the rapid transient phase of the BK-induced calcium response. That the loss of the plateau phase was due to blockage of calcium entry rather than stimulation of calcium pump activity was confirmed by examining the influx of Ba2+ following BK stimulation. The Ca2+ imaging results were confirmed by 45Ca2+ uptake measurements and extended to another tyrosine kinase inhibitor (methyl 2,5-dihydroxycinnamate), which was found to interfere with the fura-2 signal and therefore could not be used in imaging experiments. The fact that three structurally distinct inhibitors of tyrosine kinase activity inhibited BK-stimulated calcium influx, while an inactive analogue of genistein (daidzein) did not, strongly suggests the involvement of tyrosine kinases in the regulation of a BK-induced calcium entry pathway. To our knowledge, this is the first report of tyrosine kinase involvement in the regulation of calcium entry following activation of a receptor that lacks endogenous tyrosine kinase activity and is known to be coupled to phosphatidylinositol turnover. We found that calcium entry in HSWP (human foreskin fibroblast) cells can also be stimulated by emptying the intracellular calcium stores with thapsigargin. Genistein also inhibits the plateau phase of the thapsigargin-induced calcium response while leaving the transient phase intact. This suggests that the Ca2+ influx pathway induced by depletion of intracellular calcium stores with thapsigargin also may be regulated via a tyrosine kinase pathway.

Barium↗
PubMed1993

Characterization of an interleukin-2 (IL-2)-induced tyrosine phosphorylated 116-kDa protein associated with the IL-2 receptor beta-subunit.

In this paper we have extended previous results on interleukin-2 receptor (IL2-R) signal transduction and focused on the interleukin-2 (IL-2)-stimulated tyrosine phosphorylation of a 116-kDa protein (p116) observed in IL-2 responsive cells. This protein exhibited rapid and transient phosphorylation kinetics in both human T-lymphocytes and the YT cell line, attaining maximum tyrosine phosphorylation within 5 min of stimulation with IL-2. Tyrosine phosphorylated p116 co-purified with activated IL-2 receptor beta-chain (IL2-R beta) when IL2-R complexes were covalently stabilized with the membrane-permeable cleavable cross-linking agent dithiobis(succimidyl propionate) prior to detergent cell lysis and immunoprecipitation with monoclonal anti-IL2-R beta antibodies. Under these conditions comparable amounts of tyrosine-phosphorylated p116 were immunoprecipitated with either anti-IL2-R beta antibodies or anti-phosphotyrosine antibodies, suggesting that a major portion of tyrosine phosphorylated p116 is associated with the IL2-R beta subunit. Furthermore, unphosphorylated p116 was also associated with unactivated IL2-R beta, based on the observation that p116 from unstimulated YT cells underwent tyrosine phosphorylation in IL2-R beta immune-complex tyrosine kinase assay as demonstrated by anti-phosphotyrosine immunoblotting. The presence of tyrosine kinase activity in affinity-purified IL2-R beta complexes supports the notion of a preformed receptor-kinase complex. The co-association of both p116 and tyrosine kinase activity with the IL2-R beta supports the critical role of the beta-chain in IL2-R signal transduction and suggests that p116 may have a role in the dynamics of IL2-R activation.

Cell Line↗
PubMedDate not supplied

Mapping of tyrosine kinase autophosphorylation sites with synthetic peptide substrates.

Tyrosine phosphorylation is a key step in the regulation of many cellular events including signal transduction of stimulated growth factor receptor tyrosine kinases. Upon ligand activation, these proteins undergo dimerization and subsequent auto- and transphosphorylation events on specific tyrosine residues, which enables them to interact with several cellular signaling proteins. We have used synthetic peptides encompassing all the tyrosine residues of a tyrosine kinase and employed them as substrates in in vitro kinase reactions. Using this assay we have shown that short tyrosine-containing peptides derived from the cytoplasmic domain of the mouse platelet-derived growth factor beta receptor (PDGF-R) can serve as specific phosphorylation targets for the kinase. These peptides include 7 out of 8 tyrosines that are known auto- or transphosphorylation sites in vivo, as previously determined by peptide mapping and mutational analyses. We have also identified 10 additional tyrosine-containing peptides that are phosphorylated and represent possible novel auto- or transphosphorylation sites of PDGF-R. The presented method greatly simplifies the mapping of auto- or transphosphorylation sites in tyrosine kinases and provides a valuable tool in the analysis of signaling mechanisms involving these proteins.

Amino Acid Sequence↗
PubMed1995

Activation of p58c-fgr and p53/56lyn in adherent human neutrophils: evidence for a role of divalent cations in regulating neutrophil adhesion and protein tyrosine kinase activities.

Tumor necrosis factor (TNF) stimulates generation of reactive oxygen intermediates, secretion of granule constituents, and rearrangement of the cytoskeleton in neutrophils (PMN); this response requires that PMN be adherent to plasma or extracellular matrix proteins, and is dependent on beta 2 integrins. Tyrosine phosphorylation of distinct proteins [Fuortes et al., J Cell Biol 120:777-784, 1993] and activation of the protein tyrosine kinase p58c-fgr [Berton et al., J Cell Biol 126:1111-1121, 1994] were recently recognized as signals involved in beta 2 integrin-dependent responses of TNF-treated PMN. As the integrin capability to bind their ligands is regulated by divalent cations we investigated whether modulation of PMN adhesion to fibrinogen by divalent cations also affected activation of protein tyrosine kinases. In the absence of divalent cations or in the presence of Ca2+ alone, PMN did not adhere to fibrinogen in response to TNF. However, Mg2+, either alone or together with Ca2+, promoted stimulated adhesion to fibrinogen. We also found that Mn2+ promoted PMN adhesion to fibrinogen without additional stimuli. Analysis of the activity of two src family tyrosine kinases, p58c-fgr and p53/56lyn, showed that their autophosphorylating kinase activity strictly correlated with adhesion. In fact, only in the presence of Mg2+, but not in the absence of divalent cations or in the presence of Ca2+ alone, TNF increased p58c-fgr and p53/56lyn kinase activities; and this was prevented by anti-CD18 antibodies. In addition, Mn2+ strongly promoted activation of p58c-fgr and p53/56lyn without additional stimuli. Analysis of tyrosine phosphorylated proteins with anti-phosphotyrosine immunoblots showed that divalent cations regulated adhesion and protein tyrosine phosphorylation in the same fashion. Detergent extraction of proteins showed that the Mg(2+)-dependent, TNF-stimulated adhesion redistributed p58c-fgr and p53/56lyn to a Triton-insoluble fraction. In addition, analysis of p58c-fgr activity allowed us to demonstrate that the fraction of p58c-fgr which became Triton-insoluble displayed a higher kinase activity. These findings establish that PMN adhesion signals for activation of two different src family tyrosine kinases. The evidence that Mn2+, a strong promoter of integrin function, induces adhesion and activation of tyrosine kinases without additional stimuli suggest the existence of a direct link between beta 2 integrins binding to fibrinogen and activation of tyrosine kinases in neutrophils.

Antibodies↗
PubMedDate not supplied

Regulation of sperm function by protein tyrosine phosphorylation in diverse wild felid species.

Protein tyrosine phosphorylation is associated with sperm capacitation and the acrosome reaction in several mammalian species. Changes in phosphorylation of a 95-kDa protein in human, mouse, and domestic cat spermatozoa are known to be influenced by capacitation and exposure to zona pellucida (ZP) proteins. We previously reported diminished phosphorylation of 95- and 160-kDa proteins in spermatozoa from teratospermic cats, compared with normospermic domestic cats. To determine if these proteins and mechanisms are present in other species in the phenotypically diverse Felidae family, we examined the relationship between tyrosine-phosphorylated sperm proteins and sperm morphology in the leopard cat (approximately 65% normal sperm/ejaculate), tiger (approximately 65%), clouded leopard (approximately 15%), and cheetah (approximately 30%). Furthermore, we investigated the involvement of cyclic adenosine monophosphate (cAMP) in the regulation of sperm protein tyrosine phosphorylation. Specifically, we assessed the following: 1) presence of tyrosine-phosphorylated proteins in sperm extracts; 2) changes in protein tyrosine phosphorylation after sperm capacitation and swim-up separation; 3) impact of tyrosine kinase inhibition on leopard cat sperm protein phosphorylation and ZP penetration; and 4) involvement of a cAMP-dependent pathway in the regulation of protein tyrosine phosphorylation. Immunoblotting analysis with anti-phosphotyrosine antibody (PY20) indicated that a 95-kDa protein was present in all four species. Additional phosphorylated proteins were detected in the leopard cat (145- and 175-kDa proteins), tiger (185-kDa protein), clouded leopard (160- and 190-kDa proteins), and cheetah (115- and 155-kDa proteins). Sperm capacitation in vitro increased phosphorylation of one or more proteins in the leopard cat, tiger and clouded leopard, but not in the cheetah. Although swim-up separation increased the proportion of morphologically normal spermatozoa in the clouded leopard and cheetah, no changes were observed in phosphorylation of the 95-kDa sperm protein. Thus, phosphorylation of the 95-kDa protein appeared to be related to the condition of teratospermia. Exposing leopard cat spermatozoa to the tyrosine kinase inhibitor, tyrphostin, reduced (P < 0.05) phosphorylation of the 95- and 145-kDa proteins, as well as ZP penetration, without affecting sperm motility. Similarly, when spermatozoa were incubated in the presence of cAMP analogs or active and inactive stereoisomers of cAMP, phosphorylation of sperm proteins was either stimulated or inhibited. Together, these data suggest that protein tyrosine kinase mechanisms appear conserved within the family Felidae and are regulated by a cAMP/protein kinase A pathway.

Animals↗
PubMed1997

Functional analysis of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal transduction: the two YxxL segments within a single CD3zeta-ITAM are functionally distinct.

Functional analysis of the immunoreceptor tyrosine-based activation motif (ITAM) derived from the membrane-proximal ITAM of CD3zeta demonstrates that mutations at either the tyrosine or leucine residues in the N-terminal YxxL segment of the ITAM abolish all signal transduction functions of this ITAM. In contrast, mutations at the tyrosine or leucine residues in the C-terminal YxxL segment abrogate signals for interleukin (IL)-2 production but do not prevent tyrosine phosphorylation of the N-terminal tyrosine of the ITAM, lck association with the ITAM, activation of phospholipase C-gamma1 or calcium mobilization. Cross-linking of chimeric receptors containing a C-terminal YxxL leucine mutation induces tyrosine phosphorylation of ZAP70 but without stable binding to the phosphorylated ITAM. These results indicate that the two YxxL segments in an ITAM are functionally distinct and that both are essential for ZAP70 binding and IL-2 production. Furthermore, tyrosine phosphorylation of ZAP70 per se is not sufficient to trigger the downstream events leading to IL-2 production. Substitution of an alanine for the bulky side chain at the Y+1 position of the N-terminal YxxL segment reduces the receptor cross-linking requirement necessary to achieve cellular activation and the absolute dependence on lck in this process. Our results reveal that both the number of ITAM as well as the specific amino acid residues within a single ITAM determine the extent of chimeric receptor cross-linking required to trigger tyrosine phosphorylation-dependent signaling events.

Amino Acid Sequence↗
PubMed2006

Differing mechanisms of cAMP- versus seawater-induced oocyte maturation in marine nemertean worms II. The roles of tyrosine kinases and phosphatases.

Instead of blocking oocyte maturation as it does in most animals, cAMP causes oocytes of marine nemertean worms to initiate maturation (=germinal vesicle breakdown, "GVBD"). To characterize cAMP-induced GVBD in nemerteans, inhibitors of tyrosine kinase signaling were tested on Cerebratulus sp. oocytes that had been incubated in cAMP-elevating drugs versus seawater (SW) alone. Such tests yielded similar results for Src-like tyrosine kinase blockers, as the inhibitors prevented mitogen-activated protein kinase (MAPK) activation without stopping either GVBD or maturation-promoting factor (MPF) activation in both SW and cAMP-elevating treatments. Alternatively, genistein, a general tyrosine kinase antagonist, and piceatannol, an inhibitor of the tyrosine kinase Syk, reduced GVBD and MAPK/MPF activities in SW-, but not cAMP-induced maturation. Similarly, inhibitors of the human epidermal growth factor receptor-2 (HER-2) tyrosine kinase prevented GVBD and MAPK/MPF activations in oocytes treated with SW, but not with cAMP-elevating drugs. Antagonists of either protein tyrosine phosphatases (PTPs) or the dual-specificity phosphatase Cdc25 also reduced GVBD and MAPK/MPF activities in SW-treated oocytes without generally affecting cAMP-induced maturation. Collectively, these data suggest cAMP triggers GVBD via pathways that do not require MAPK activation or several components of tyrosine kinase signaling. In addition, such differences in tyrosine kinase cascades, coupled with the dissimilar patterns of Ser/Thr kinase signaling described in the accompanying study, indicate that nemertean oocytes are capable of utilizing multiple mechanisms to activate MPF during GVBD.

Animals↗
PubMed2005

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery.

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H2O2, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase alpha chain is probably tyrosine-phosphorylated, but demonstrated that the beta chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.

Animals↗
PubMed1994

Insulin stimulates the phosphorylation of Tyr538 and the catalytic activity of PTP1C, a protein tyrosine phosphatase with Src homology-2 domains.

PTP1C is a non-transmembrane protein-tyrosine phosphatase and contains two Src homology-2 (SH2) domains. Insulin stimulated the tyrosine phosphorylation of PTP1C in human 1M-9 lymphoblast cells, in rat H35 hepatoma cells and in Chinese hamster ovary cells over-expressing both insulin receptors and PTP1C. Insulin also stimulated the tyrosine phosphorylation of a mutant PTP1C lacking SH2 domains in Chinese hamster ovary cells, suggesting that the SH2 domains are not required for insulin-stimulated tyrosine phosphorylation of PTP1C. The insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1C in a cell-free system. Peptide mapping of phosphorylated PTP1C showed that Tyr538 in the C-terminal region was phosphorylated in response to insulin. The tyrosine phosphorylation of PTP1C by the insulin receptor kinase increased phosphatase activity. Furthermore, PTP1C was shown to bind to autophosphorylated insulin receptors through its C-terminal region, but PTP1C did not bind to unphosphorylated receptors. These results suggest that PTP1C is a target protein for the insulin receptor tyrosine kinase and that the C-terminal region of PTP1C may function both in the regulation of phosphatase activity and in the association of PTP1C with autophosphorylated insulin receptors.

Amino Acid Sequence↗