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PubMed2000

Analysis of FAK-associated signaling pathways in the regulation of cell cycle progression.

Focal adhesion kinase (FAK) is an important mediator of signal transduction pathways initiated by integrins in cell migration, survival and cell cycle regulation. The ability of FAK to mediate integrin signaling in the regulation of cell cycle progression depends on the phosphorylation of Tyr397, which implies a functional significance for the formation of FAK signaling complexes with Src, phosphatidylinositol-3-kinase (PI3K) and Grb7. We have previously described a FAK mutant, D395A, that selectively disrupts FAK binding to PI3K, but allows FAK association with Src. Using this mutation in a mislocalized FAK mutant background, we show here that formation of a FAK/PI3K complex is not sufficient for cell cycle progression but the formation of a FAK/Src complex plays an essential role. We also show that mutation of D395 to A disrupted FAK association with Grb7. This suggests that a FAK/Grb7 complex is not involved in the cell cycle regulation either, which is supported by direct analysis of cells expressing a dominant negative Grb7 construct. Finally, we provide evidence that the Src-dependent association of FAK with Grb2 and p130(Cas) are both required for the regulation of cell cycle progression by FAK. Together, these studies identify important FAK downstream signaling pathways in cell cycle regulation.

Adaptor Proteins, Signal Transducing↗

PubMed2004

A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase.

A direct binding site for the Grb2 adapter protein is required for the induction of fatal chronic myeloid leukemia (CML)-like disease in mice by Bcr-Abl. Here, we demonstrate direct binding of Grb2 to the Tel-Abl (ETV6-Abl) fusion protein, the product of complex (9;12) chromosomal translocations in human leukemia, via tyrosine 314 encoded by TEL exon 5. A Tel-Abl point mutant (Y314F) and a splice variant without TEL exon 5 sequences (Deltae5) lacked Grb2 interaction and exhibited decreased binding and phosphorylation of the scaffolding protein Gab2 and impaired activation of phosphatidylinositol 3-kinase, Akt, and extracellular signal-regulated kinase/mitogen-activated protein kinase in hematopoietic cells. Tel-Abl Y314F and Deltae5 were unable to transform fibroblasts to anchorage-independent growth and were defective for B-lymphoid transformation in vitro and lymphoid leukemogenesis in vivo. Previously, we demonstrated that full-length Tel-Abl induced two distinct myeloproliferative diseases in mice: CML-like leukemia similar to that induced by Bcr-Abl and a novel syndrome of small-bowel myeloid infiltration endotoxemia and hepatic and renal failure. Lack of the Grb2 binding site had no effect on development of small bowel syndrome but significantly attenuated the induction of CML-like disease by Tel-Abl. These results suggest that direct binding of Grb2 is a common mechanism contributing to leukemogenesis by oncogenic Abl fusion proteins.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Oxidant-induced hypertrophy of A549 cells is accompanied by alterations in eukaryotic translation initiation factor 4E and 4E-binding protein-1.

Control of protein synthesis resides at the level of eukaryotic translation initiation (eIF) complex formation. Complex formation is regulated by the mRNA cap-binding protein, eIF4E, whose activity is influenced by phosphorylation and binding to 4E-binding protein 1 (4E-BP1). To provide a link between alterations in protein synthesis and the pathogenesis of oxidant-mediated lung disease, we investigated the effect of hydrogen peroxide (H2O2) on actively growing A549 cells. Cells were exposed to 200 or 400 microM H2O2 for 4 h and then assessed for changes in proliferation, protein synthesis, and eIF4E and 4E-BP1 status over 72 h. We found that both concentrations of H2O2 inhibited [3H]thymidine incorporation and cell division while inducing a G2/M-predominant growth arrest within 24 h. In addition, H2O2 increased cell size, [3H]leucine incorporation/cell, and total cell protein. Although time had little effect on eIF4E and 4E-BP1 expression and phosphorylation state of control cells, H2O2 induced a 2- to 3-fold increase in eIF4E and 4E-BP1 expression, a 5-fold increase in eIF4E phosphorylation, and a shift in the distribution of 4E-BP1 phosphorylation favoring lesser phosphorylated forms. These findings suggest that oxidant-mediated alterations in protein synthesis and cell morphology occur in concert with changes in factors known to regulate translation kinetics.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Grb2 forms an inducible protein complex with CD28 through a Src homology 3 domain-proline interaction.

CD28 provides a costimulatory signal that results in optimal activation of T cells. The signal transduction pathways necessary for CD28-mediated costimulation are presently unknown. Engagement of CD28 leads to its tyrosine phosphorylation and subsequent binding to Src homology 2 (SH2)-containing proteins including the p85 subunit of phosphatidylinositol 3'-kinase (PI3K); however, the contribution of PI3K to CD28-dependent costimulation remains controversial. Here we show that CD28 is capable of binding the Src homology 3 (SH3) domains of several proteins, including Grb2. The interaction between Grb2 and CD28 is mediated by the binding of Grb2-SH3 domains to the C-terminal diproline motif present in the cytoplasmic domain of CD28. While the affinity of the C-terminal SH3 domain of Grb2 for CD28 is greater than that of the N-terminal SH3 domain, optimal binding requires both SH3 domains. Ligation of CD28, but not tyrosine-phosphorylation, is required for the SH3-mediated binding of Grb2 to CD28. We propose a model whereby the association of Grb2 with CD28 occurs via an inducible SH3-mediated interaction and leads to the recruitment of tyrosine-phosphorylated proteins such as p52(shc) bound to the SH2 domain of Grb2. The inducible interaction of Grb2 to the C-terminal region of CD28 may form the basis for PI3K-independent signaling through CD28.

Adaptor Proteins, Signal Transducing↗

PubMed1993

Exocytosis of vacuolar apical compartment (VAC) in Madin-Darby canine kidney epithelial cells: cAMP is involved as second messenger.

Vacuolar apical compartment (VAC) is a transient organelle originally observed in Madin-Darby canine kidney (MDCK) epithelial cells impaired from forming cell-cell contacts. VACs are large vacuoles which contain microvilli and apical plasma membrane markers (among others, a 184-kDa plasma membrane protein, AP2), but exclude basolateral membrane markers. Upon reestablishment of cell-cell contacts, VACs are rapidly (within 1 h) exocytosed toward intercellular spaces, after which the apical plasma membrane drifts toward its final destination (Vega-Salas, Salas, and Rodriguez-Boulan. 1988. J. Cell Biol. 107, 1717-1728). In this work, we studied the role of cAMP as a mediator for the exocytosis of VACs. We shifted confluent cells from low to normal calcium medium (thus reestablishing cell-cell contacts and causing VAC exocytosis), a shift which resulted in a significant rise of cellular levels of both total intracellular and protein-bound cAMP. The 8-Br analog of cAMP (8-Br-cAMP) (5-50 microM) caused externalization of the intracellular compartment of AP2 as measured by radioimmunoassay. A similar effect was observed with 3-isobutyl-1-methylxanthine. 8-Br-cAMP also caused the appearance of AP2-positive VAC images in nonpermeabilized cells, namely, VACs that become accessible to extracellular antibodies upon fusion with the plasma membrane. Lanthanum, which abolishes the peak of intracellular free calcium during a calcium switch, failed to block the exocytosis. On the other hand, 12-O-tetradecanoylphorbol-13-acetate induced only a modest exocytic response. Finally, 8-Br-cAMP induced VAC exocytosis in sparse MDCK cells grown in normal calcium medium. These data indicate that cAMP is a mediator between the extracellular signal provided by cell-cell contacts and VAC exocytosis.

1-Methyl-3-isobutylxanthine↗

PubMed2003

Formation of a tumour necrosis factor receptor 1 molecular scaffolding complex and activation of apoptosis signal-regulating kinase 1 during seizure-induced neuronal death.

The consequences of activation of tumour necrosis factor receptor 1 (TNFR1) during neuronal injury remain controversial. The apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, can mediate cell death downstream of TNFR1. Presently, we examined the formation of the TNFR1 signalling cascade and response of ASK1 during seizure-induced neuronal death. Brief (40 min) seizures were induced in rats by intra-amygdala microinjection of kainic acid, which elicited unilateral hippocampal CA3 neuronal death. Seizures caused a rapid decline in the expression of the silencer of death domains protein within injured CA3. Co-immunoprecipitation analysis revealed a commensurate assembly of a TNFR1 scaffold complex containing TNFR-associated death domain protein, receptor interacting protein and TNFR-activating factor 2. In addition, recruitment of TNFR-activating factor 2 was likely promoted by Bcl10-mediated sequestering of cellular inhibitor of apoptosis protein 2. Apoptosis signal-regulating kinase 1 was sequestered in a complex that contained the molecular chaperone 14-3-3beta and protein phosphatase 5. Seizures triggered its dissociation, and the phosphorylation of the ASK1 substrates, mitogen-activated protein kinase kinase 3/6 and 4. Subsequently, protein phosphatase 5 translocated into the nuclei of degenerating CA3 neurons, while ASK1 colocalized with the adaptor proteins Daxx and TNFR-activating factor 2 at the outer membrane of injured CA3 neurons. Neutralizing antibodies to TNFalpha reduced the numbers of DNA damaged cells within the injured hippocampus. These data suggest ASK1 may be involved in the mechanism of seizure-induced neuronal death downstream of a TNFR1 death-signalling complex.

14-3-3 Proteins↗

PubMed1998

Regulation of adipocyte gene expression by polyunsaturated fatty acids.

A wide number of adipocyte genes are regulated by exogenous polyunsaturated fatty acids (PUFA) through the actions of the peroxisome proliferator activated receptor. Such genes include the adipocyte lipid-binding protein (ALBP or aP2) which plays a central role in facilitating the trafficking of fatty acids within adipocytes. Work from a number of laboratories has suggested the key elements of the lipid signal transduction pathway include: (1) the transport of exogenous PUFAs across the plasma membrane, (2) metabolism of polyunsaturated fatty acids to second messengers including 15-deoxy delta 12,14 prostaglandin J2 (15dPGJ2), (3) trafficking of 15dPGJ2 and other second messengers from the smooth ER to the nucleus for association with peroxisome proliferator activated receptor gamma (PPAR gamma), and (4) dimerization of PPAR gamma with retinoid X receptor (RXR) permitting regulation of transcription via association with any of several nuclear co-activators or repressors. In addition to the aP2 gene being a target of activation by fatty acids, at the protein level ALBP/aP2 plays a role in trafficking of fatty acids and/or their metabolises. We report here that in a heterologous system using CV-1 cells transiently transfected with PPAR gamma 2, co-expression of ALBP/aP2 enhances the PPAR-dependent activation of gene transcription. These results suggest that ALBP/aP2 functions as a positive factor in fatty acid signalling by directly targetting and delivering fatty acids metabolites to the lipid signal transduction pathway.

Adaptor Protein Complex 2↗

PubMed2005

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I.

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The polarity-establishment component Bem1p interacts with the exocyst complex through the Sec15p subunit.

Spatial regulation of the secretory machinery is essential for the formation of a new bud in Saccharomyces cerevisiae. Yet, the mechanisms underlying cross-talk between the secretory and the cell-polarity-establishment machineries have not been fully elucidated. Here, we report that Sec15p, a subunit of the exocyst complex, might provide one line of communication. Not only is Sec15p an effector of the rab protein Sec4p, the master regulator of post-Golgi trafficking, but it also interacts with components of the polarity-establishment machinery. We have demonstrated a direct physical interaction between Sec15p and Bem1p, a protein involved in the Cdc42p-mediated polarity-establishment pathway, confirming a prior two-hybrid study. When this interaction is compromised, as in the case of cells lacking the N-terminal 138 residues of Bem1p, including the first Src-homology 3 (SH3) domain, the localization of green fluorescent protein (GFP)-tagged Sec15 is affected, especially in the early stage of bud growth. In addition, Sec15-1p, which is defective in Bem1p binding, mislocalizes along with Sec8p, another exocyst subunit. Overall, our evidence suggests that the interaction of Sec15p with Bem1p is important for Sec15p localization at the early stage of bud growth and, through this interaction, Sec15p might play a crucial role in integrating the signals between Sec4p and the components of the early-polarity-establishment machinery. This, in turn, helps to coordinate the secretory pathway and polarized bud growth.

Adaptor Proteins, Signal Transducing↗

PubMed2004

PP2A phosphatase activity is required for stress and Tor kinase regulation of yeast stress response factor Msn2p.

In response to stress and nutrient starvation, the Saccharomyces cerevisiae transcription factor Msn2p accumulates in the nucleus and activates expression of a broad array of genes. Here, we analyze the role of the Tor (target of rapamycin) signaling pathway in mediating these responses. Inactivation of the Tor pathway component Tap42p using tap42(Ts) alleles causes a sustained nuclear localization similar to that after the addition of the Tor kinase inhibitor rapamycin. Effects of Tap42p inactivation and rapamycin addition could be suppressed by deletion of TIP41, which encodes a Tap42p-interacting protein. These results support the notion that rapamycin affects Msn2p by inactivating Tap42p function. Tap42p interacts with the catalytic subunit of PP2A (protein phosphatase 2A) and PP2A-like phosphatases. Deletion of either the catalytic or regulatory subunit that forms the PP2A phosphatase complex prevents nuclear accumulation of Msn2p in the tap42(Ts) strain and in wild-type strains treated with rapamycin. These results suggest that Tap42p is an inhibitor of PP2A phosphatase, which in turn inhibits nuclear export of Msn2p. Interestingly, PP2A function is also required for nuclear accumulation of Msn2p in response to stresses, such as heat and osmotic shock, as well as nitrogen (but not glucose) starvation. Thus, PP2A and the Tor kinase pathway transduce stress and nitrogen starvation signals to Msn2p. Finally, Msn2p localization is unaffected by conditional loss of 14-3-3 protein function, ruling out the possibility that 14-3-3 proteins act as a scaffold to sequester Msn2p in the cytoplasm.

14-3-3 Proteins↗

PubMed2001

Recruitment of the class II phosphoinositide 3-kinase C2beta to the epidermal growth factor receptor: role of Grb2.

Previously we demonstrated that the class II phosphoinositide 3-kinase C2beta (PI3K-C2beta) is rapidly recruited to a phosphotyrosine signaling complex containing the activated receptor for epidermal growth factor (EGF). Although this association was shown to be dependent upon specific phosphotyrosine residues present on the EGF receptor, the underlying mechanism remained unclear. In this study the interaction between PI3K-C2beta and the EGF receptor is competitively attenuated by synthetic peptides derived from each of three proline-rich motifs present within the N-terminal region of the PI3K. Further, a series of N-terminal PI3K-C2beta fragments, truncated prior to each proline-rich region, bound the receptor with decreased efficiency. A single proline-rich region was unable to mediate receptor association. Finally, an equivalent N-terminal fragment of PI3K-C2alpha that lacks similar proline-rich motifs was unable to affinity purify the activated EGF receptor from cell lysates. Since these findings revealed that the interaction between the EGF receptor and PI3K-C2beta is indirect, we sought to identify an adaptor molecule that could mediate their association. In addition to the EGF receptor, PI3K-C2beta(2-298) also isolated both Shc and Grb2 from A431 cell lysates. Recombinant Grb2 directly bound PI3K-C2beta in vitro, and this effect was reproduced using either SH3 domain expressed as a glutathione S-transferase (GST) fusion. Interaction with Grb2 dramatically increased the catalytic activity of this PI3K. The relevance of this association was confirmed when PI3K-C2beta was isolated by coimmunoprecipitation with anti-Grb2 antibody from numerous cell lines. Using immobilized, phosphorylated EGF receptor, recombinant PI3K-C2beta was only purified in the presence of Grb2. We conclude that proline-rich motifs within the N terminus of PI3K-C2beta mediate the association of this enzyme with activated EGF receptor and that this interaction involves the Grb2 adaptor.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Interactions between AMPA receptors and intracellular proteins.

alpha-Amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors mediate most fast excitatory synaptic transmission in the mammalian CNS. They play a central role in synapse stabilisation and plasticity and their prolonged activation is potently neurotoxic. Developmental and activity-dependent changes in the functional synaptic expression of these receptors are subject to tight cellular regulation. The molecular and cellular mechanisms which control the postsynaptic insertion and arrangement of individual AMPA receptor variants are therefore the subject of intense investigation and in the last two years there has been significant progress towards elucidating some of the processes involved. Much of the new information has come from the application of the yeast two-hybrid assay, which has led to the discovery of a hitherto unexpected complexity of proteins which selectively interact with individual AMPA receptor subunits. These proteins have been implicated in the regulation of AMPA receptor post-translational modification, targeting and trafficking, surface expression and anchoring. The aim of this article is to present an overview of the major interacting proteins described so far and to place these in the context of how they may participate in the well ordered series of events controlling the cell biology of AMPA receptors.

Adaptor Proteins, Signal Transducing↗

PubMed1994

PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.

Phosphatidylinositol 3-kinase (PI 3-kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein-serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N-terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3-kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter-subunit serine phosphorylation in the regulation of the PI 3-kinase in vivo.

Animals↗

PubMed2003

The genetics of inflammatory bowel disease.

The inflammatory bowel diseases (IBD) comprise complex genetic disorders, with multiple contributing genes. Linkage studies have implicated several genomic regions as likely containing IBD susceptibility genes, with some observed uniquely in Crohn's disease (CD) or ulcerative colitis (UC), and others common to both disorders. The best replicated linkage region, IBD1, on chromosome 16q contains the CD susceptibility gene, NOD2/CARD15. NOD2/CARD15 is expressed in peripheral blood monocytes and is structurally related to the plant R proteins, which mediate host resistance to microbial pathogens. Three major coding region polymorphisms within NOD2/CARD15 have been highly associated with CD among patients of European descent. Having one copy of the risk alleles confers a 2-4-fold risk for developing CD, whereas double-dose carriage increases the risk 20-40-fold. All 3 major CD variants exhibit a deficit in NF-kappaB activation in response to bacterial components. Carriage of NOD2/CARD15 risk alleles is associated with ileal location, earlier disease onset, and stricturing phenotype. Other IBD genomic regions include IBD2 on chromosome 12q (observed more in UC), and IBD3, containing the major histocompatibility complex region. A short genomic region has been associated with CD on chromosome 5q, but the precise contributing gene is as yet unidentified. The characterization of additional IBD susceptibility genes could potentially lead to the identification of novel therapeutic agents for IBD, make possible a molecular reclassification of disease, and increase understanding of the contribution of environmental factors (notably, tobacco and the intestinal microbial milieu) to intestinal inflammation.

Animals↗

PubMed2001

Identification of a phospholipase C-gamma1 (PLC-gamma1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-gamma1 and NFAT.

SLP-76 is an adapter protein required for T-cell receptor (TCR) signaling. In particular, TCR-induced tyrosine phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1), and the resultant TCR-inducible gene expression, depend on SLP-76. Nonetheless, the mechanisms by which SLP-76 mediates PLC-gamma1 activation are not well understood. We now demonstrate that SLP-76 directly interacts with the Src homology 3 (SH3) domain of PLC-gamma1. Structure-function analysis of SLP-76 revealed that each of the previously defined protein-protein interaction domains can be individually deleted without completely disrupting SLP-76 function. Additional deletion mutations revealed a new, 67-amino-acid functional domain within the proline-rich region of SLP-76, which we have termed the P-1 domain. The P-1 domain mediates a constitutive interaction of SLP-76 with the SH3 domain of PLC-gamma1 and is required for TCR-mediated activation of Erk, PLC-gamma1, and NFAT (nuclear factor of activated T cells). The adjacent Gads-binding domain of SLP-76, also within the proline-rich region, mediates inducible recruitment of SLP-76 to a PLC-gamma1-containing complex via the recruitment of both PLC-gamma1 and Gads to another cell-type-specific adapter, LAT. Thus, TCR-induced activation of PLC-gamma1 entails the binding of PLC-gamma1 to both LAT and SLP-76, a finding that may underlie the requirement for both LAT and SLP-76 to mediate the optimal activation of PLC-gamma1.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Elevated connexin-43 expression in term human myometrium correlates with elevated c-Jun expression and is independent of myometrial estrogen receptors.

Just previous to the onset of parturition, a number of genes such as the one that codes for connexin-43 (Cx43) gap junction protein are induced in the myometrium. We have shown previously that activation of protein kinase C in human myometrial cultured cells leads to an up-regulation of cx43 transcription through an activating protein-1 element in the 5'-flanking promoter. Analyses were now performed on extracts of term myometrial tissue to test for an association between the up-regulation of cx43 expression and the expression of transcription factors and steroid hormone receptors that might regulate cx43 expression at term. Immunoblot analyses were performed on extracts of term myometrial tissue from women receiving elective or indicated cesarean sections to test for an association between the up-regulation of cx43 expression and the up-regulation of expression of the transcription factors c-Jun, c-Fos, and Sp1, which have cognate binding elements in the cx43 5'-flanking promoter. Immunoblot analysis, immunohistochemistry, and receptor binding assays were also performed to analyze the levels of progesterone receptors (PR) and estrogen receptors (ER) in the same term myometrial tissue, and these were compared to the levels in nonpregnancy myometrial tissue. The levels of PR were consistently 2- to 3-fold higher in term myometrial tissue than in nonpregnancy values and did not fluctuate during the menstrual cycle as did ER levels. Surprisingly, in term myometrium, ER was barely detectable by immunoblot and had whole cell diffuse staining by immunohistochemistry. In addition, very low levels of estrogen binding were observed in the term myometrial tissue. Treatment of primary myometrial cultures containing ER with estrogen for 3 or 48 h did not result in up-regulation of c-Jun or c-Fos proteins or in trans-activation from the proximal cx43 promoter with the activating protein-1 element. In contrast, an activated form of c-Jun protein was 10- to 18-fold higher in term myometrial tissue that also had elevated cx43 expression compared to c-Jun levels in term myometrial tissue with low cx43 expression. Likewise, c-Fos and Sp1 levels were 2-4 fold higher in term myometrial tissue with elevated cx43 expression. Although c-Fos and Sp1 proteins could be detected by immunoblot in myometrial tissue from nonpregnant women, c-Jun and Cx43 proteins could not. In summary, these results suggest that up-regulation of human myometrial cx43 gene expression at term involves induction of primarily c-jun expression through a mechanism that does not directly involve myometrial ER or the loss of PR. Peptide hormones that activate protein kinase cascades, such as the protein kinase C cascade, may be important to signal the onset of labor in humans.

Adaptor Protein Complex alpha Subunits↗

PubMed2005

Expression of a novel beta adaptin subunit mRNA splice variant in human testes.

AIM: To identify a novel isoform of adaptin 2 beta subunit (named Ap2beta-NY) and to investigate its relationship with testicular development and spermatogenesis. METHODS: Using a human testis cDNA microarray, a clone (Ap2beta-NY), which was strongly expressed in adult testes but weakly expressed in embryo testes, was sequenced and analyzed. Using polymerase chain reaction (PCR), the tissue distribution and expression time pattern of Ap2beta-NY were determined. RESULTS: Ap2beta-NY was identified and has been deposited in the GenBank (AY341427). The expression level of Ap2beta-NY in the adult testis was about 3-fold higher than that in the embryo testis. PCR analysis using multi-tissue cDNA indicated that Ap2beta-NY was highly expressed in the testis, spleen, thymus, prostate, ovary, blood leukocyte and brain, but not in the heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. In addition, Ap2beta-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome, which implied that, in the testis, Ap2beta-NY was restrictively expressed in germ cells. CONCLUSION: Ap2beta-NY is an isoform of Ap2beta and may be involved in regulating the process of spermatogenesis and testis development.

Adaptor Protein Complex beta Subunits↗

PubMed1995

A membrane proximal domain of the human interleukin-3 receptor beta c subunit that signals DNA synthesis in NIH 3T3 cells specifically binds a complex of Src and Janus family tyrosine kinases and phosphatidylinositol 3-kinase.

The high affinity human interleukin-3 receptor is a heterodimeric protein consisting of an alpha and beta c subunit. The beta c subunit is responsible for receptor signal transduction. We have shown that a membrane proximal domain of the cytoplasmic tail of the human beta c subunit (amino acids 451-517) is minimally required for human IL-3 to signal DNA synthesis in quiescent transfected NIH 3T3 cells. Glutathione S-transferase (GST) fusion proteins of this 451-517 region and another region 451-562 that includes an acidic domain previously shown in other receptors to bind Src family kinases were constructed. Purified Lyn and Lck kinase, but not Fes, could phosphorylate tyrosines in both domains. Adsorption with lysates from the human IL-3-dependent hematopoietic cell line (TF-1) or 3T3 cells and in vitro phosphorylation showed that both these domains were intensely phosphorylated. Phosphoamino acid analysis, however, revealed that the majority of phosphorylation was on serine and threonine rather than tyrosine. Adsorption of these domains with 3T3 or TF-1 cell lysates, followed by immunoblotting, showed that cytoplasmic tyrosine kinases Lyn, Fes, and JAK-2 could also stably associate with both domains; however, Src family kinases are more strongly recognized by both regions than JAK-2 kinase. In addition, phosphatidylinositol 3-kinase from cell lysates was also found stably associated with these domains, but GTPase activating protein, Vav, Sos1, or Grb2 were not.

3T3 Cells↗