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Query: UNIPROT:P06889 (Mol)
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Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.
Mol Biol Cell 1995 Dec
PMID:Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions. 859 Aug

The dynamic instability of microtubules is thought to be regulated by MAPs and phosphorylation. Here we describe the effect of the neuronal microtubule-associated protein tau by observing the dynamics of single microtubules by video microscopy. We used recombinant tau isoforms and tau mutants, and we phosphorylated tau by the neuronal kinases MARK (affecting the KXGS motifs within tau's repeat domain) and cdk5 (phosphorylating Ser-Pro motifs in the regions flanking the repeats). The variants of tau can be broadly classified into three categories, depending on their potency to affect microtubule dynamics. "Strong" tau variants have four repeats and both flanking regions. "Medium" variants have one to three repeats and both flanking regions. "Weak" variants lack one or both of the flanking regions, or have no repeats; with such constructs, microtubule dynamics is not significantly different from that of pure tubulin. N- or C-terminal tails of tau have no influence on dynamic instability. The two ends of microtubules (plus and minus) showed different activities but analogous behavior. These results are consistent with the "jaws" model of tau where the flanking regions are considered as targeting domains whereas the addition of repeats makes them catalytically active in terms of microtubule stabilization. The dominant changes in the parameters of dynamic instability induced by tau are those in the dissociation rate and in the catastrophe rate (up to 30-fold). Other rates change only moderately or not at all (association rate increased up to twofold, rates of rescue or rapid shrinkage decreased up to approximately twofold). The order of repeats has little influence on microtubule dynamics (i.e., repeats can be re-arranged or interchanged), arguing in favor of the "distributed weak binding" model proposed by Butner and Kirschner (1991); however, we confirmed the presence of a "hotspot" of binding potential involving Lys274 and Lys281 observed by Goode and Feinstein, 1994. Phosphorylation of Ser-Pro motifs by cdk5 (mainly Ser 202, 235, and 404) in the flanking regions had a moderate effect on microtubule dynamics while phosphorylation at the "Alzheimer"-site Ser262 MARK eliminated tau's interactions with microtubules. In both cases the predominant effects of phosphorylation are on the rates of tubulin dissociation and catastrophe whereas the effects on the rates of association or rescue are comparatively small.
Mol Biol Cell 1995 Dec
PMID:Domains of tau protein, differential phosphorylation, and dynamic instability of microtubules. 859 Aug 13

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.
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PMID:Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants. 862 39

Two chimeric receptors, ER1 and ER2, were constructed. ER1 contains the extracellular and transmembrane (TM) domains derived from epidermal growth factor receptor and the cytoplasmic domain from c-Ros; ER2 is identical to ER1 except that its TM domain is derived from c-Ros. Both chimeras can be activated by epidermal growth factor and are capable of activating or phosphorylating an array of cellular signaling proteins. Both chimeras promote colony formation in soft agar with about equal efficiency. Surprisingly, ER1 inhibits while ER2 stimulates cell growth on monolayer culture. Cell cycle analysis revealed that all phases, in particular the S and G2/M phases, of the cell cycle in ER1 cells were elongated whereas G1 phase of ER2 cells was shortened threefold. Comparison of signaling pathways mediated by the two chimeras revealed several differences. Several early signaling proteins are activated or phosphorylated to a higher extent in ER1 than in ER2 cells in response to epidermal growth factor. ER1 is less efficiently internalized and remains tyrosine phosphorylated for a longer time than ER2. However, phosphorylation of the 66-kDa She protein, activation of mitogen activated protein kinase, and induction of c-fos and c-jun occur either to a lesser extent or for a shorter time in ER1 cells. Cellular protein phosphorylation patterns are also different in ER1 and ER2 cells. In particular, a 190-kDa Shc-associated protein is tyrosine phosphorylated in ER2 but not in ER1 cells. Our results indicate that the TM domains have a profound effect on the signal transduction and biological activity of those chimeric receptors. The results also imply that sustained stimulation of ER1 due to its retarded internalization apparently triggers an inhibitory response that dominantly counteracts the receptor-mediated mitogenic signals. These two chimeras, expressed at similar levels in the same cell type but having opposite effects on cell growth, provide an ideal system to study the mechanism by which a protein tyrosine kinase inhibits cell growth.
Mol Cell Biol 1996 Apr
PMID:Two chimeric receptors of epidermal growth factor receptor and c-Ros that differ in their transmembrane domains have opposite effects on cell growth. 865 24

A true protein kinase CKII (CKII) activity was characterized in liver mitochondria by its phosphorylating activity on the specific peptide substrate of CKII, the binding and elution profile of the enzyme on a phosphocellulose column and immunostaining of a 36 kDa polypeptide with antibodies against the alpha-subunit of human CKII. This CKII activity was located predominantly in the intermembrane space of quiescent mitochondria. A translocation of the enzyme to inner membrane of energized mitochondria occurred in the presence of spermine. Translocated CKII activity was tightly bound to inner membrane, and high salt concentrations were necessary to release the activity. The inner face of the inner membrane could constitute the in vivo localization of mitochondrial CKII since the potential substrates of the enzyme are 4 matrix proteins.
Cell Mol Biol (Noisy-le-grand) 1996 Mar
PMID:Evidence of true protein kinase CKII activity in mitochondria and its spermine-mediated translocation to inner membrane. 869 55

In yeast the GCN2 kinase mediates translational control of GCN4 by phosphorylating the alpha subunit of eIF-2 in response to extracellular amino acid limitation. Although phosphorylation of eIF-2 alpha has been shown to inhibit global protein synthesis, amino acid starvation results in a specific activation effect on GCN4 mRNA translation. Under the same conditions, translation of other mRNAs appears only slightly affected. The mechanism responsible for the observed selectivity of the GCN2 kinase is not clear. Here, we present genetic evidence that suggests that locally restricted action of the GCN2 kinase facilitates GCN4-specific translational regulation.
Mol Gen Genet 1996 Jul 19
PMID:Genetic evidence for functional specificity of the yeast GCN2 kinase. 870 69

Short term (15 min) effects of activators of protein kinase A (PKA), PKC and PKG on cardiac macroscopic (g(j)) and single channel (gamma j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell voltage-clamp, PKC activation by 100 nM TPA increased g(j) by 16 +/- 2% (mean +/- S.E.M, n = 9), 1.5 mM of the PKG activator 8-bromo-cGMP (8Br-cGMP) decreased g(j) by 26 +/- 2% (n = 4), whereas 1.5 mM of the PKA activator 8Br-cAMP did not affect g(j) (1 +/- 5%, n = 11). Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two gamma j sizes of 20 pS and 40-45 pS. Under control conditions, the larger events were most frequently observed. Whereas 8Br-cAMP did not change this distribution, TPA or 8Br-cGMP shifted the gamma j distribution to the lower sizes. Diffusion of 6-carboxyfluorescein (6-CF), a gap junction permeant tracer, from the injected cell to neighboring cells was studied on small clusters of neonatal rat cardiomyocytes. Under control conditions, 6-CF labeled 8.4 +/- 0.4 cells (mean +/- S.E.M, n = 31). Whereas 8Br-cAMP did not change the extent of dye transfer (8.1 +/- 0.5 cells, n = 10), TPA restricted the diffusion of 6-CF to 2.2 +/- 0.2 cells (n = 30) and 8Br-cGMP to 3.5 +/- 0.3 cells (n = 10). This suggests that permeability and single channel conductance of Cx43 gap junction channels are parallel related. Altogether, these results point to the differential modulation of electrical and metabolic coupling of cardiac cells by various phosphorylating conditions.
Mol Cell Biochem
PMID:Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions. 873 33

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.
Mol Cell Biol 1996 Sep
PMID:In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation. 875 31

G protein-coupled receptors activate phospholipase C (PLC)-beta isoforms by the alpha or beta gamma subunits of G proteins, whereas growth-factor receptors activate PLC-gamma isoforms by phosphorylating tyrosine residues of the enzyme. As a common substrate for PLC enzymes, phosphatidylinositol 4,5-bisphosphate [Ptdins(4,5)P2] may play a pivotal role in the regulation of cellular PLC activity. Because small-molecular-weight G proteins have been implicated in the synthesis of Ptdins(4,5)P2, we studied the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates small G proteins of the Rho family, on receptor-stimulated PLC activity. We report here that in N1E-115 neuroblastoma cells, stimulation of inositol phosphate formation by the G protein-coupled receptor agonists bradykinin and lysophosphatidic acid and by the tyrosine kinase receptor agonist platelet-derived growth factor is largely attenuated by toxin B treatment. Furthermore, inositol phosphate production stimulated by the stable GTP analog guanosine 5'-O-(3-thio)-triphosphate in permeabilized N1E-115 cells was inhibited by C3 exoenzyme, which specifically inactivates Rho proteins. The inhibition by toxin B was apparently not caused by its effect on the cytoskeleton. In addition, the level of platelet-derived growth factor receptors, which was studied with immunoblotting, was unaffected by toxin B. Using exogenous Ptdlns(4,5)P2 as PLC substrate, it was found that the intrinsic enzymatic activity of PLC activated either by Ca2+ or by guanosine 5'-O-(3-thio)triphosphate was not altered by toxin B. However, toxin B decreased strongly, by up to 80%, the cellular level of Ptdins(4,5)P2 in a concentration-dependent manner, without changing those of phosphatidylinositol and phosphatidylinositol 4-phosphate. These results, together with the recent finding that Rho family proteins can regulate phosphatidylinositol 4-phosphate 5-kinase activity, demonstrate that Rho proteins are presumably important regulators of Ptdins(4,5)P2 synthesis and, thereby, play an integral role in the regulation of cellular signaling by PLC enzymes.
Mol Pharmacol 1996 Oct
PMID:Inhibition by toxin B of inositol phosphate formation induced by G protein-coupled and tyrosine kinase receptors in N1E-115 neuroblastoma cells: involvement of Rho proteins. 886 31

Transcription factor, cAMP response element-binding protein (CREB), which is phosphorylated by cAMP-dependent kinase via an increase in cAMP, and regulates gene transcription by binding to the cAMP response element (CRE) on target genes. We examined age-dependent alterations in the DNA-binding activity of CREB in rat brain regions, and the effects of rolipram, a cAMP-specific phosphodiesterase (PDE) inhibitor on the CRE-binding activity by electrophoretic mobility-shift assay (EMSA). A marked age-dependent decrease in the CRE-binding activity was shown in all brain regions examined, especially in the basal forebrain, the striatum and the hippocampus. Furthermore, CRE-binding activities in the basal forebrain of both young-adult and aged rats significantly increased 2 h after rolipram administration (1 mg/kg, i.p.), and the rolipram treatment recovered the decreased CRE-binding activity in the aged rats. The saturation experiment in EMSA also revealed that rolipram reversed the decrease in the maximum CRE-bindings in the basal forebrain with aging. Since the 5' upstream region of the rat choline acetyltransferase (ChAT) gene contains CRE, and ChAT-positive neurons in the basal forebrain project to the frontal cortex and the hippocampus, rolipram may exert its previously reported ameliorating effect on the age-related reductions of ChAT activities in the frontal cortex and the hippocampus by phosphorylating CREB in the basal forebrain with activation of cAMP-dependent protein kinase via inhibition of PDE.
Brain Res Mol Brain Res 1996 Sep 05
PMID:Alterations of cAMP response element-binding activity in the aged rat brain in response to administration of rolipram, a cAMP-specific phosphodiesterase inhibitor. 888 54


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