Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation

Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase implicated in cell death and smooth muscle contractility, but its mechanism of regulation is unknown. We have identified six phosphorylation sites in ZIPK that regulate both its enzyme activity and localization, including Thr180, Thr225, Thr265, Thr299, Thr306, and Ser311. Mutational analysis showed that phosphorylation of Thr180 in the kinase activation T-loop, Thr225 in the substrate-binding groove, and Thr265 in kinase subdomain X is essential for full ZIPK autophosphorylation and activity toward exogenous substrates. Abrogation of phosphorylation of Thr299, Thr306, and Ser311 had little effect on enzyme activity, but mutation of Thr299 and Thr300 to alanine resulted in redistribution of ZIPK from the cytosol to the nucleus. Mutation of Thr299 alone to alanine caused ZIPK to assume a diffuse cellular localization, whereas T299D redistributed the enzyme to the cytoplasm. C-terminal truncations of ZIPK at amino acid 273 or 342 or mutation of the leucine zipper motif increased ZIPK activity toward exogenous substrates by severalfold, suggesting a phosphorylation-independent autoinhibitory role for the C-terminal domain. Additionally, mutation of the leucine zipper reduced the ability of ZIPK to oligomerize and also caused ZIPK to relocalize from the cytoplasm to the nucleus in vivo. Together, our findings show that ZIPK is positively regulated by phosphorylation within its kinase domain and that it contains an inhibitory C-terminal domain that controls enzyme activity, localization, and oligomerization.     

Regulation of the insulin receptor kinase by hyperinsulinism

A murine fibroblast cell line transfected with human insulin receptor cDNA, NIH 3T3 HIR3.5, was observed to display insulin-induced down-regulation of insulin-binding activity in a time- and concentration-dependent manner. Maximal inhibition of insulin-binding activity (54%) occurred within 16 h of exposure to 100 nM insulin in vivo, where in vivo refers to intact cells in tissue culture. The decrease in cellular insulin-binding activity was the consequence of a decrease in the number of cell-associated insulin receptors as determined by Scatchard analysis of insulin binding, 125I-insulin affinity cross-linking, and Western blotting of the insulin receptor beta subunit. Acute insulin treatment in vivo (1-60 min) resulted in the activation of the insulin receptor protein tyrosine kinase as determined by in vitro phosphorylation of glutamic acid:tyrosine (4:1), where in vitro refers to broken cell preparations. This acute in vivo insulin activation of the insulin receptor tyrosine kinase resulted in a greater stimulation (1.4-1.9-fold) of tyrosine kinase activity in the glutamic acid:tyrosine (4:1) assay than the maximal stimulation produced by insulin treatment in vitro. In contrast, long term (24 h) insulin treatment in vivo resulted in a 50-70% decrease in intrinsic protein tyrosine kinase activity of the insulin receptors compared with that of acutely activated (1 min) insulin receptors. Under these conditions, the insulin receptor protein kinase activity remained insulin independent in the in vitro substrate kinase assay. Surprisingly, the insulin-independent activated (1 min in vivo insulin-treated) and uncoupled (24 h in vivo insulin-treated) insulin receptors displayed similar stoichiometries of 32P incorporation into the beta subunit by in vitro autophosphorylation when compared with the control insulin receptors, ranging from 1.5 to 1.8 mol of phosphate incorporated/mol of insulin receptor. Phosphoamino acid analysis demonstrated that the phosphoserine/phosphothreonine content of in vivo 32P-labeled insulin receptors increased markedly within a 1-h exposure to insulin in vivo, whereas insulin-induced receptor desensitization was not apparent until 10-24 h after exposure to insulin. These data suggest that insulin treatment in vivo results initially in the activation of the insulin receptor kinase followed by a subsequent uncoupling of protein kinase activity. This insulin-induced desensitization of the insulin receptor kinase does not correlate with the extent of beta subunit serine/threonine phosphorylation.     

Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) via a phorbol ester/PKC-dependent pathway involves phosphorylation events. The present study identifies five in vivo phosphorylation sites by mass spectrometry, and the role of four of them was investigated by site-directed mutagenesis. Four sites are autophosphorylation sites, the first of which (Ser(916)) is located in the C terminus; its phosphorylation modifies the conformation of the kinase and influences duration of kinase activation but is not required for phorbol ester-mediated activation of PKD. The second autophosphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3tau. The last two autophosphorylation sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the full-length PKD; they may affect enzyme catalysis but are not involved in the activation of wild-type PKD by phorbol ester. We also present evidence for proteolytic activation of PKD. The fifth site (Ser(255)) is transphosphorylated downstream of a PKC-dependent pathway after in vivo stimulation with phorbol ester. In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very distinct ways.     

Regulation of insulin receptor kinase activity by insulin mimickers and an insulin antagonist

Three agents which mimic insulin action in intact cells (concanavalin A, wheat germ agglutinin, and polyclonal insulin receptor antibody), mimicked insulin's ability to stimulate the kinase activity of purified insulin receptors. In contrast, monoclonal insulin receptor antibody, an antagonist of insulin action, did not stimulate the phosphorylation of the insulin receptor either in intact IM-9 cells or in purified receptor preparations. This antibody, however, antagonized the ability of insulin to stimulate the phosphorylation of the receptor both in intact cells and in the purified receptor. These studies with insulin mimickers and an insulin antagonist are consistent with a role for the kinase activity of the receptor mediating the actions of insulin.     

Regulation of AMP-activated protein kinase by multisite phosphorylation in response to agents that elevate cellular cAMP

The AMP-activated protein kinase (AMPK) and cAMP signaling systems are both key regulators of cellular metabolism. In this study, we show that AMPK activity is attenuated in response to cAMP-elevating agents through modulation of at least two of its alpha subunit phosphorylation sites, viz. alpha-Thr(172) and alpha1-Ser(485)/alpha2-Ser(491), in the clonal beta-cell line INS-1 as well as in mouse embryonic fibroblasts and COS cells. Forskolin, isobutylmethylxanthine, and the glucose-dependent insulinotropic peptide inhibited AMPK activity and reduced phosphorylation of the activation loop alpha-Thr(172) via inhibition of calcium/calmodulin-dependent protein kinase kinase-alpha and -beta, but not LKB1. These agents also enhanced phosphorylation of alpha-Ser(485/491) by the cAMP-dependent protein kinase. AMPK alpha-Ser(485/491) phosphorylation was necessary but not sufficient for inhibition of AMPK activity in response to forskolin/isobutylmethylxanthine. We show that AMPK alpha-Ser(485/491) can be a site for autophosphorylation, which may play a role in limiting AMPK activation in response to energy depletion or other regulators. Thus, our findings not only demonstrate cross-talk between the cAMP/cAMP-dependent protein kinase and AMPK signaling modules, but also describe a novel mechanism by which multisite phosphorylation of AMPK contributes to regulation of its enzyme activity.     

Ca2+/Calmodulin-dependent protein kinase kinase beta is regulated by multisite phosphorylation

Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a serine/threonine-directed kinase that is activated following increases in intracellular Ca(2+). CaMKKβ activates Ca(2+)/calmodulin-dependent protein kinase I, Ca(2+)/calmodulin-dependent protein kinase IV, and the AMP-dependent protein kinase in a number of physiological pathways, including learning and memory formation, neuronal differentiation, and regulation of energy balance. Here, we report the novel regulation of CaMKKβ activity by multisite phosphorylation. We identify three phosphorylation sites in the N terminus of CaMKKβ, which regulate its Ca(2+)/calmodulin-independent autonomous activity. We then identify the kinases responsible for these phosphorylations as cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). In addition to regulation of autonomous activity, we find that phosphorylation of CaMKKβ regulates its half-life. We find that cellular levels of CaMKKβ correlate with CDK5 activity and are regulated developmentally in neurons. Finally, we demonstrate that appropriate phosphorylation of CaMKKβ is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKKβ-dependent signaling cascades.     

The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase

Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulin's ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.     

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

src kinase catalyzes the phosphorylation and activation of the insulin receptor kinase

When a partially purified insulin receptor preparation immobilized on insulin-agarose is incubated with [gamma-32P]ATP, Mn2+, and Mg2+ ions, the receptor beta subunit becomes 32P-labeled. The 32P-labeling of the insulin receptor beta subunit is increased by 2-3-fold when src kinase is included in the phosphorylation reaction. In addition, the presence of src kinase results in the phosphorylation of a Mr = 125,000 species. The Mr = 93,000 receptor beta subunit and the Mr = 125,000 32P-labeled bands are absent when an insulin receptor-deficient sample, prepared by the inclusion of excess free insulin to inhibit the adsorption of the receptor to the insulin-agarose, is phosphorylated in the presence of the src kinase. These results indicate that the insulin receptor alpha and beta subunits are phosphorylated by the src kinase. The src kinase-catalyzed phosphorylation of the insulin receptor is not due to the activation of receptor autophosphorylation because a N-ethylmaleimide-treated receptor preparation devoid of receptor kinase activity is also phosphorylated by the src kinase. Conversely, the insulin receptor kinase does not catalyze phosphorylation of the active or N-ethylmaleimide-inactivated src kinase. Subsequent to src kinase-mediated tyrosine phosphorylation, the insulin receptor, either immobilized on insulin-agarose or in detergent extracts, exhibits a 2-fold increase in associated kinase activity using histone as substrate. src kinase mediates phosphorylation of predominantly tyrosine residues on both alpha and beta subunits of the insulin receptor. Tryptic peptide mapping of the 32P-labeled receptor alpha and beta subunits by high pressure liquid chromatography reveals that the src kinase-mediated phosphorylation sites on both receptor subunits exhibit elution profiles identical with those phosphorylated by the receptor kinase. Furthermore, the HPLC elution profile of the receptor auto- or src kinase-catalyzed phosphorylation sites on the receptor alpha subunit are also identical with that on the receptor beta subunit. These results indicate that: the src kinase catalyzes tyrosine phosphorylation of the insulin receptor alpha and beta subunits; and src kinase-catalyzed phosphorylation of insulin receptor can mimic the action of autophosphorylation to activate the insulin receptor kinase in vitro, although whether this occurs in intact cells remains to be determined.     

Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1.

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.     

Tyrosine phosphorylation of pp185 by insulin receptor kinase in a cell-free system

Insulin treatment of rat H-35 hepatoma cells causes rapid tyrosine phosphorylation of a high molecular weight protein termed pp185 besides autophosphorylation of the beta-subunit of the insulin receptor (IR) in an intact cell system. To elucidate the molecular basis for tyrosine phosphorylation of pp185, cell-free phosphorylation of pp185 was performed using phosphotyrosine-containing proteins (PYPs) purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibody. After insulin treatment of cells, marked increases of tyrosine phosphorylation of pp185 and IR were observed compared to noninsulin-treated cells. Site-specific antibodies that specifically inactivate IR kinase inhibited tyrosine phosphorylation of pp185 as well as the beta-subunit of IR. PYPs purified from detergent-free cell extracts contained pp185 but little IR; tyrosine phosphorylation of pp185 did not occur. Addition of IR kinase purified from human placenta to these PYPs restored insulin-dependent tyrosine phosphorylation of pp185. These results suggest that tyrosine phosphorylation of pp185 is catalyzed directly by IR kinase in this cell-free system.     

Serine phosphorylation of insulin receptor substrate 1 by inhibitor kappa B kinase complex

Insulin resistance contributes importantly to the pathophysiology of type 2 diabetes mellitus. One mechanism mediating insulin resistance may involve the phosphorylation of serine residues in insulin receptor substrate-1 (IRS-1), leading to impairment in the ability of IRS-1 to activate downstream phosphatidylinositol 3-kinase-dependent pathways. Insulin-resistant states and serine phosphorylation of IRS-1 are associated with the activation of the inhibitor kappaB kinase (IKK) complex. However, the precise molecular mechanisms by which IKK may contribute to the development of insulin resistance are not well understood. In this study, using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser(307) (equivalent to Ser(312) in human IRS-1), we observed serine phosphorylation of IRS-1 in response to TNF-alpha or calyculin A treatment that paralleled surrogate markers for IKK activation. The phosphorylation of human IRS-1 at Ser(312) in response to tumor necrosis factor-alpha was significantly reduced in cells pretreated with the IKK inhibitor 15 deoxy-prostaglandin J(2) as well as in cells derived from IKK knock-out mice. We observed interactions between endogenous IRS-1 and IKK in intact cells using a co-immunoprecipitation approach. Moreover, this interaction between IRS-1 and IKK in the basal state was reduced upon IKK activation and increased serine phosphorylation of IRS-1. Data from in vitro kinase assays using recombinant IRS-1 as a substrate were consistent with the ability of IRS-1 to function as a direct substrate for IKK with multiple serine phosphorylation sites in addition to Ser(312). Taken together, our data suggest that IRS-1 is a novel direct substrate for IKK and that phosphorylation of IRS-1 at Ser(312) (and other sites) by IKK may contribute to the insulin resistance mediated by activation of inflammatory pathways.     

Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.     

Regulation of heart insulin receptor tyrosine kinase activity by magnesium and spermine

Insulin action and aspects of the insulin-signaling pathway have been studied in the heart although the direct regulation of the heart’s insulin receptor has not been explored. This study describes the first purification and characterization of the mammalian (rabbit, rat and bovine) heart insulin receptor. The rabbit heart IR showed maximum insulin binding of 18 μg/mg (~1 mole insulin/mole (α₂β₂) receptor) and a curvilinear Scatchard plot with a high affinity K_D for insulin binding of ~4 nM at optimal pH (7.8) and NaCl concentration (150 mM). The insulin receptor tyrosine kinase activity was stimulated by insulin, Mg²⁺ (half-maximum response at ~5.6–10.6 nM and ~8.5 mM, respectively) and by the physiological polyamines, spermine and spermidine. The stimulation by Mg²⁺ and the polyamines occurred with and without insulin. These characteristics of the heart insulin receptor provide a mechanism for regulating the activity of the receptor’s tyrosine kinase activity by the intracellular free Mg²⁺ concentration and the polyamines in the absence and presence of insulin.     

Identification of major tyrosine phosphorylation sites in the human insulin receptor substrate Gab-1 by insulin receptor kinase in vitro

Gab-1 (Grb2-associated binder-1), which appears to play a central role in cellular growth response, transformation, and apoptosis, is a member of the insulin receptor substrate (IRS) family. IRS proteins act downstream in the signaling pathways of different receptor tyrosine kinases, including the insulin receptor (IR). In this paper, we characterize the phosphorylation of recombinant human Gab-1 (hGab-1) by IR in vitro. Kinetic phosphorylation data revealed that hGab-1 is a high affinity substrate for the IR (K(M): 12.0 microM for native IR vs 23.3 microM for recombinant IR). To elucidate the IR-specific phosphorylation pattern of hGab-1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Phosphorylated tyrosine residues were subsequently identified by sequencing the separated phosphopeptides by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that hGab-1 was phosphorylated by IR at eight tyrosine residues (Y242, Y285, Y373, Y447, Y472, Y619, Y657, and Y689). Seventy-five percent of the identified radioactivity was incorporated into tyrosine residues Y447, Y472, and Y619 exhibiting features (NYVPM motif) of potential binding sites for the regulatory subunit (p85) of phosphatidylinositol (PI)-3 kinase. Accordingly, pull down assays with human HepG2 cell lysates showed that IR-specific phosphorylation of wild-type hGab-1 strongly enhanced PI-3 kinase binding. This is still the case when a single tyrosine residue in the NYVPM motif was mutated to phenylalanine. In contrast, phosphorylation-dependent binding of PI-3 kinase was completely abolished by changing a second tyrosine residue in a NYVPM motif independent from its location. Recently, we identified a similar cohort of tyrosine phosphorylation sites for the epidermal growth factor receptor (EGFR) with a predominant phosphorylation of tyrosine residue Y657 and binding of Syp [Lehr, S. et al. (1999) Biochemistry 38, 151-159]. These differences in the phosphorylation pattern of hGab-1 may contribute to signaling specificity by different tyrosine kinase receptors engaging distinct SH2 signaling molecules.     

Inhibition of insulin receptor phosphorylation by indomethacin

Insulin stimulated phosphorylation of tyrosine residues by the insulin receptor kinase may be part of a signalling mechanism associated with insulin's action. We report that indomethacin inhibited the phosphorylation of the -subunit of the solubilized adipocyte insulin receptor. Indomethacin also inhibited several insulin-sensitive processes in intact rat adipocytes. Indomethacin (1 mM) inhibited basal phosphorylation of the -subunit of the solubilized insulin receptor by 6007o and insulin-stimulated phosphorylation by 30%. In adipocytes, indomethacin inhibited basal 3-0-[methyl-¹⁴C]-methyl-D glucose transport by 50070 (P < 0.01), D-[6-¹⁴C]-glucose oxidation by 5007o (P < 0.01), D-[6-¹⁴C]-glucose conversion to lipid by 30010 (P < 0.01), and D-[1-¹⁴C]-glucose conversion to lipid by 6007o (P<0.01). Similarly, indomethacin inhibited insulin-stimulated 3-0-[methyl-¹⁴C]-methyl-D-glucose transport by 75070 (P<0.01), D-[6-¹⁴C]-glucose oxidation by 20% (P<0.05), D-[1-¹⁴C]-glucose oxidation by 35070 (P<0.01), D-[6-¹⁴C] glucose conversion to lipid by 25010 (P<0.01), and D-[1-¹⁴C] glucose conversion to lipid by 4501o (P<0.01). In contrast, insulin binding to its receptor, basal D-[1-¹⁴C]-glucose oxidation and both basal and insulin-stimulated activation of glycogen synthase were unaffected by indomethacin. Thus, indomethacin partially inhibited autophosphorylation of the solubilized insulin receptor on tyrosine and partially inhibited some but not all of insulin's actions. This supports the hypothesis that insulin's metabolic effects are linked to activation of the insulin receptor protein kinase and indicates that there may be heterogeneity in the mechanisms of intracellular metabolic control by insulin.     

Regulation of the phosphorylation of the inositol 1,4,5-trisphosphate receptor by protein kinase C

The various inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms are potential substrates for several protein kinases. We compared the in vitro phosphorylation of purified IP(3)R1 and IP(3)R3 by the catalytic subunit of protein kinase C (PKC). Phosphorylation of IP(3)R1 by PKC was about eight times stronger than that of IP(3)R3 under identical conditions. Protein kinase A strongly stimulated the PKC-induced phosphorylation of IP(3)R1. In contrast, Ca(2+) inhibited its phosphorylation (IC(50)相似文献   

Differential regulation of a CLC anion channel by SPAK kinase ortholog-mediated multisite phosphorylation

Shrinkage-induced inhibition of the Caenorhabditis elegans cell volume and cell cycle-dependent CLC anion channel CLH-3b occurs by concomitant phosphorylation of S742 and S747, which are located on a 175 amino acid linker domain between cystathionine-β-synthase 1 (CBS1) and CBS2. Phosphorylation is mediated by the SPAK kinase homolog GCK-3 and is mimicked by substituting serine residues with glutamate. Type 1 serine/threonine protein phosphatases mediate swelling-induced channel dephosphorylation. S742E/S747E double mutant channels are constitutively inactive and cannot be activated by cell swelling. S742E and S747E mutant channels were fully active in the absence of GCK-3 and were inactive when coexpressed with the kinase. Both channels responded to cell volume changes. However, the S747E mutant channel activated and inactivated in response to cell swelling and shrinkage, respectively, much more slowly than either wild-type or S742E mutant channels. Slower activation and inactivation of S747E was not due to altered rates of dephosphorylation or dephosphorylation-dependent conformational changes. GCK-3 binds to the 175 amino acid inter-CBS linker domain. Coexpression of wild-type CLH-3b and GCK-3 with either wild-type or S742E linkers gave rise to similar channel activity and regulation. In contrast, coexpression with the S747E linker greatly enhanced basal channel activity and increased the rate of shrinkage-induced channel inactivation. Our findings suggest the intriguing possibility that the phosphorylation state of S742 in S747E mutant channels modulates GCK-3/channel interaction and hence channel phosphorylation. These results provide a foundation for further detailed studies of the role of multisite phosphorylation in regulating CLH-3b and GCK-3 activity.     

Regulation of plant symbiosis receptor kinase through serine and threonine phosphorylation

We studied the biochemical properties of a plant receptor-like kinase to gain insights into the regulatory mechanism of this largest class of plant kinases. SYMRK (symbiosis receptor kinase) is required for early signal transduction leading to plant root symbioses with nitrogen-fixing rhizobia and phosphate-acquiring arbuscular mycorrhizal fungi. Amino acid substitutions in positions critical for activity of other related kinases cause a nonsymbiotic plant phenotype, suggesting that SYMRK kinase activity is required for symbiosis. SYMRK is capable of intermolecular autophosphorylation. Nonphosphorylated SYMRK is less active than the phosphorylated version, suggesting the phosphorylation status of SYMRK determines its activity. Three Ser/Thr residues were identified as residues required for full kinase activation through targeted mutagenesis. Using quadrupole time-of-flight mass spectrometry analysis, two of these were confirmed to be phosphorylated in vitro. These crucial phosphorylation sites are conserved among various plant receptor-like kinases as well as animal Pelle/interleukin-1 receptor associated kinase. Despite the distinct domain architecture of receptor-like kinases versus Pelle/interleukin-1 receptor associated kinase, our results suggest the existence of conserved activation mechanisms.