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PubMed1996

Identification of the G protein-coupled receptor kinase phosphorylation sites in the human beta2-adrenergic receptor.

Rapid desensitization of G protein-coupled receptors is mediated, at least in part, by their phosphorylation by the G protein-coupled receptor kinases (GRKs). However, only in the case of rhodopsin have the actual sites of receptor phosphorylation been unambiguously determined. Although previous studies have implicated the cytoplasmic tail of the beta2-adrenergic receptor (beta2AR) as the site of GRK-mediated phosphorylation, the identities of the phosphorylated residues were unknown. Here we report the identification of the sites of GRK2- and GRK5-mediated beta2AR phosphorylation. The phosphorylation sites of both serine/threonine kinases reside exclusively in a 40-amino acid peptide located at the extreme carboxyl terminus of the beta2AR. Of the seven phosphorylatable residues within this peptide, six are phosphorylated by GRK5 (Thr-384, Thr-393, Ser-396, Ser-401, Ser-407, and Ser-411) and four are phosphorylated by GRK2 (Thr-384, Ser-396, Ser-401, and Ser-407) at equivalent phosphorylation stoichiometries (approximately 1.0 mol Pi/mol receptor). In addition to the GRK5-specific phosphorylation of Thr-393 and Ser-411, differences in the distribution of phosphate between sites are observed for GRK2 and GRK5. Increasing the stoichiometry of GRK2-mediated beta2AR phosphorylation from approximately 1.0 to 5.0 mol Pi/mol receptor increases the stoichiometry of phosphorylation of Thr-384, Ser-396, Ser-401, and Ser-407 rather than increasing the number of phosphoacceptor sites. The location of multiple GRK2 and GRK5 phosphoacceptor sites at the extreme carboxyl terminus of the beta2AR is highly reminiscent of GRK1-mediated phosphorylation of rhodopsin.

Amino Acid Sequence↗
PubMed1997

Casein kinase II catalyzes tyrosine phosphorylation of the yeast nucleolar immunophilin Fpr3.

In the yeast Saccharomyces cerevisiae, the nucleolar immunophilin, Fpr3, is phosphorylated at tyrosine and dephosphorylated by the phosphotyrosine-specific phosphoprotein phosphatase, Ptp1. In Ptp1-deficient cells, Fpr3 contains phospho-Tyr at a single site (Tyr184), but also contains phospho-Ser and phospho-Thr. Ser186 (adjacent to Tyr184) is situated within a canonical site for phosphorylation by casein kinase II (CKII). Yeast cell lysates contain an activity that binds to Fpr3 in vitro and phosphorylates Fpr3 at Ser, Thr, and Tyr; this activity was found to be dependent on expression of functional yeast CKII. Moreover, purified Fpr3 was phosphorylated on Tyr184 in vitro by either purified yeast CKII or purified, bacterially-expressed human CKII. Likewise, phosphorylation of Fpr3 at tyrosine in vivo was markedly enhanced in yeast cells overexpressing a heterologous (Drosophila) CKII, but was undetectable in yeast cells carrying only a temperature-sensitive allele of the endogenous CKII, even when the cells were grown at a permissive temperature. Phosphorylation of Fpr3 at Tyr184 by CKII in vitro lagged behind phosphorylation of Fpr3 at Ser, and was accelerated by pre-phosphorylation of Fpr3 at Ser using CKII. Furthermore, synthetic peptides corresponding to the sequence surrounding Tyr184 that contained P-Ser (or Glu) at position 186 were much more efficient substrates for CKII phosphorylation of Tyr184 than a synthetic peptide containing Ala at position 186. These findings indicate that CKII phosphorylates Fpr3 at tyrosine and serine both in vivo and in vitro and thus possesses dual specificity. These results also indicate that Tyr184 is phosphorylated by CKII via a two-step process, in which phosphorylation at the +2 position provides a negatively-charged specificity determinant that allows subsequent phosphorylation of Tyr184.

Amino Acid Isomerases↗
PubMed1997

Regulation of the epithelial brush border Na+/H+ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger.

The epithelial brush border Na+/H+ exchanger isoform 3 (NHE3) is regulated by growth factors and protein kinases. When stably expressed in PS120 fibroblasts, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by phorbol esters. To examine the role of phosphorylation of NHE3 in growth factor/protein kinase regulation, NHE3 was C-terminally tagged with an 11-amino acid epitope of the vesicular stomatitis virus glycoprotein (VSVG) and stably expressed in Na+/H+ exchanger null PS120 fibroblasts (PS120/NHE3V). NHE3V was regulated by serum, FGF, and phorbol ester in a manner identical to wild type non-VSVG-tagged NHE3. Phosphorylation of NHE3V was evaluated via immunoprecipitation with anti-VSVG antibody after in vivo labeling of PS120/NHE3V cells with [32P]orthophosphate. NHE3V was phosphorylated under basal conditions. However, FGF and PMA, under conditions in which these agonists regulate NHE3V, altered neither the amount of phosphorylation of NHE3V as analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography nor two-dimensional phosphopeptide maps of tryptic digests of NHE3V. In contrast, while changes in NHE3V phosphorylation were not observed with serum exposure by one-dimensional SDS-polyacrylamide gel electrophoresis, two-dimensional studies showed increases in two phosphopeptides. Under all these conditions, phosphoamino acid analysis showed that NHE3V was phosphorylated only on serine residues. By cell surface protein biotinylation studies under basal conditions, at least 27% of the NHE3V was expressed on the cell surface. To further analyze the phosphorylation status of the surface and intracellular forms of NHE3V under basal conditions and determine whether the amount of phosphorylation of the surface form changes upon serum, FGF, and PMA regulation, the surface form of NHE3V was separated from intracellular form by biotinylation/avidin-agarose precipitation. Under basal conditions, both intracellular and surface forms of NHE3V were phosphorylated. However, the amount of phosphorylation of the surface form of NHE3V did not change upon stimulation by serum and FGF and inhibition by PMA based on one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography. Thus, we conclude that when expressed in PS120 cells, while NHE3 is a phosphoprotein under basal conditions, its regulation by FGF and PMA is not by changes in the phosphorylation of NHE3, while regulation by serum may involve changes in its phosphorylation. Regulation of NHE3 probably involves intermediate associated regulatory proteins. The function of basal phosphorylation of NHE3 is not known.

Animals↗
PubMed1997

Phosphorylation by neuronal cdc2-like protein kinase promotes dimerization of Tau protein in vitro.

In Alzheimer's disease, the microtubule-associated protein tau forms paired helical filaments (PHFs) that are the major structural component of neurofibrillary tangles. Although tau isolated from PHFs (PHF-tau) is abnormally phosphorylated, the role of this abnormal phosphorylation in PHF assembly is not known. Previously, neuronal cdc2-like protein kinase (NCLK) was shown to phosphorylate tau on sites that are abnormally phosphorylated in PHF-tau (Paudel, H. K., Lew, J., Ali, Z., and Wang, J. H. (1993) J. Biol. Chem. 268, 23512-23518). In this study, phosphorylation by NCLK was found to promote dimerization of recombinant human tau (R-tau) and brain tau (B-tau) purified from brain extract. Chemical cross-linking by disuccinimidyl suberate (DSS), a homobifunctional chemical cross-linker that specifically cross-linked R-tau dimers, and a Superose 12 gel filtration chromatography revealed that R-tau preparations contain mixtures of monomeric and dimeric R-tau species. When the structure of NCLK-phosphorylated R-tau was studied by a similar approach, DSS preferentially cross-linked the phosphorylated R-tau over the nonphosphorylated R-tau, and the phosphorylated R-tau eluted as a dimeric species from the gel filtration column. Phosphorylated R-tau became resistant to DSS upon dephosphorylation and was recovered as a monomeric species from the gel filtration column. In the presence of a low concentration of dithiothreitol (1.65 microM), R-tau formed disulfide cross-linked R-tau dimers. When compared, phosphorylated R-tau formed more disulfide cross-linked dimers than the nonphosphorylated R-tau. B-tau also was specifically cross-linked to dimers by DSS. When B-tau and NCLK-phosphorylated B-tau were treated with DSS, phosphorylated B-tau was preferentially cross-linked over nonphosphorylated counterpart. Taken together, these results suggest that phosphorylation by NCLK promotes dimerization and formation of disulfide cross-linked tau dimers, which is suggested to be the key step leading to PHF assembly (Schweers, O., Mandelkow, E.-M., Biernat, J., and Mandelkow, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8463-8467).

Animals↗
PubMed1998

Mechanisms involved in the acidosis enhancement of the isoproterenol-induced phosphorylation of phospholamban in the intact heart.

Previous experiments have shown that acidosis enhances isoproterenol-induced phospholamban (PHL) phosphorylation (Mundiña-Weilenmann, C., Vittone, L., Cingolani, H. E., Orchard, C. H. (1996) Am. J. Physiol. 270, C107-C114). In the present experiments, performed in isolated Langendorff perfused rat hearts, phosphorylation site-specific antibodies to PHL combined with the quantitative measurement of 32P incorporation into PHL were used as experimental tools to gain further insight into the mechanism involved in this effect. At all isoproterenol concentrations tested (3-300 nM), phosphorylation of Thr17 of PHL was significantly higher at pHo 6.80 than at pHo 7.40, without significant changes in Ser16 phosphorylation. This increase in Thr17 phosphorylation was associated with an enhancement of the isoproterenol-induced relaxant effect. In the absence of isoproterenol, the increase in [Ca]o at pHo 6.80 (but not at pHo 7.40) evoked an increase in PHL phosphorylation that was exclusively due to an increase in Thr17 phosphorylation and that was also associated with a significant relaxant effect. This effect and the phosphorylation of Thr17 evoked by acidosis were both offset by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. In the presence of isoproterenol, either the increase in [Ca]o or the addition of a 1 microM concentration of the phosphatase inhibitor okadaic acid was able to mimic the increase in isoproterenol-induced Thr17 phosphorylation produced by acidosis. In contrast, these two interventions have opposite effects on phosphorylation of Ser16. Whereas the increase in [Ca]o significantly decreased phosphorylation of Ser16, the addition of okadaic acid significantly increased the phosphorylation of this residue. The results are consistent with the hypothesis that the increase in phospholamban phosphorylation produced by acidosis in the presence of isoproterenol is the consequence of two different mechanisms triggered by acidosis: an increase in [Ca2+]i and an inhibition of phosphatases.

Acidosis↗
PubMed1998

Protein kinase C-dependent tyrosine phosphorylation of p130cas in differentiating neuroblastoma cells.

The cell signaling docking protein p130cas became tyrosine-phosphorylated in SH-SY5Y human neuroblastoma cells during induced differentiation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum or a combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The differentiating cells develop a neuronal phenotype with neurites and growth cones and sustained activation of protein kinase C (PKC) and pp60c-src. The TPA-induced p130cas phosphorylation increased within 5 min of stimulation and persisted for at least 4 days, whereas bFGF/IGF-I-induced p130cas phosphorylation was biphasic. However, the increase in tyrosine phosphorylation of p130cas was not restricted to differentiation inducing stimuli. The phosphorylation was blocked by the specific PKC inhibitor GF 109203X, and transient transfection with active PKC-epsilon induced p130cas tyrosine phosphorylation. pp60c-src, known to directly phosphorylate p130cas in other cell systems, was not activated after stimulation with TPA or bFGF/IGF-I for up to 30 min, and the initial p130cas phosphorylation was resistant to the Src family kinase inhibitor herbimycin A. However, in long term stimulated cells, herbimycin A blocked the induced phosphorylation of p130cas. Also, overexpression of src induced phosphorylation of p130cas. p130cas protein and phosphorylated p130cas were present in growth cones isolated from differentiated SH-SY5Y cells. Inhibition of PKC activity in differentiating cells with GF 109203X leads to a rapid retraction of growth cone filopodia, and p130cas phosphorylation decreased transiently (within minutes). Growth cones isolated from these cells were virtually devoid of phosphorylated p130cas. These data suggest a function for p130cas as a PKC downstream target in SH-SY5Y cells and possibly also in their growth cones.

Bucladesine↗
PubMed2001

Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton.

We have previously shown that tyrosine phosphorylation of the actin-regulatory protein villin is accompanied by the redistribution of phosphorylated villin and a concomitant decrease in the F-actin content of intestinal epithelial cells. The temporal and spatial correlation of these two events suggested that tyrosine phosphorylation of villin may be involved in the rearrangement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetics of actin polymerization by reconstituting in vitro the tyrosine phosphorylation of villin and its association with actin. Full-length recombinant human villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding properties of villin were examined using a co-sedimentation assay. Phosphorylation of villin did not change the stoichiometry (1:2) but decreased the binding affinity (4.4 microm for unphosphorylated versus 0.6 microm for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we demonstrated that tyrosine phosphorylation had a negative effect on actin nucleation by villin. In contrast, tyrosine phosphorylation enhanced actin severing by villin. Electron microscopic analysis showed complementary morphological changes. Phosphorylation inhibited the actin bundling and enhanced the actin severing functions of villin. Taken together our data show that tyrosine phosphorylation of villin decreases the amount of villin bound to actin filaments, inhibits the actin-polymerizing properties of villin, and promotes the actin-depolymerizing functions instead. These observations suggest a role for tyrosine phosphorylation in modulating the microvillar cytoskeleton in vivo by villin in response to specific physiological stimuli.

Actins↗
PubMed2000

Phosphorylation and regulation of a Gq/11-coupled receptor by casein kinase 1alpha.

Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1alpha (CK1alpha). In the current study we investigate the involvement of CK1alpha in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1alpha, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by approximately 40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser(345)-Leu(463)) is an inhibitor of CK1alpha due to its ability to both act as a pseudo-substrate for CK1alpha and form a high affinity complex with CK1alpha. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1alpha is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an approximately 80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4, 5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response.

Animals↗
PubMed2001

Regulation of photoreceptor phosphodiesterase (PDE6) by phosphorylation of its inhibitory gamma subunit re-evaluated.

Phosphorylation of the inhibitory gamma subunit (Pgamma) of rod cGMP phosphodiesterase (PDE6) has been reported to turn off visual excitation without the requirement for inactivation of the photoreceptor G-protein transducin. We evaluated the significance of Pgamma phosphorylation for PDE6 regulation by preparing Pgamma stoichiometrically phosphorylated at Thr(22) or at Thr(35). Phosphorylation of Pgamma at either residue caused a minor decrease--not the previously reported increase--in the ability of Pgamma to inhibit catalysis at the active site of purified PDE6 catalytic dimers. Likewise, Pgamma phosphorylation had little effect on its potency to inhibit transducin-activated PDE6 depleted of its endogenous Pgamma subunits. The strength of Pgamma interaction with the regulatory GAF domain of PDE6 was reduced severalfold upon Pgamma phosphorylation at Thr(22) (but not Thr(35)), as judged by allosteric changes in cGMP binding to these noncatalytic sites on the enzyme (Mou, H., and Cote, R. H. (2001) J. Biol. Chem. 276, 27527-27534). In contrast, the effects of Pgamma phosphorylation on its interactions with activated transducin were much more pronounced. Phosphorylation of Pgamma at either Thr(22) or Thr(35) greatly diminished its ability to bind activated transducin, consistent with earlier work. In situ phosphorylation of Pgamma by endogenous rod outer segment kinases was enhanced severalfold upon light activation, but only approximately 10% of the endogenous Pgamma was phosphorylated. This is attributed to Pgamma being a poor substrate for protein kinases when associated with the PDE6 holoenzyme. We conclude that, contrary to previous reports, Pgamma phosphorylation at either Thr(22) or Thr(35) modestly weakens its direct interactions with PDE6. However, Pgamma phosphorylation subsequent to its dissociation from PDE6 is likely to abolish its binding to activated transducin and may serve to make phosphorylated Pgamma available to regulate other signal transduction pathways (e.g. mitogen-activated protein kinase; Wan, K. F., Sambi, B. S., Frame, M., Tate, R., and Pyne, N. J. (2001) J. Biol. Chem. 276, 37802-37808) in photoreceptor cells.

Animals↗
PubMed2001

Partial phosphorylation of the N-formyl peptide receptor inhibits G protein association independent of arrestin binding.

It is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin.

Amino Acid Sequence↗
PubMed2001

Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase.

Na(+)/H(+) exchanger regulatory factor (NHERF)-1 is a PDZ domain-containing adaptor protein known to bind to various receptors, channels, cytoskeletal elements, and cytoplasmic signaling proteins. We report here that the phosphorylation state of NHERF-1 is profoundly regulated by the cell cycle: NHERF-1 in HeLa cells is hyperphosphorylated in mitosis phase and much less phosphorylated at other points of the cell cycle. This mitosis phase-dependent phosphorylation of NHERF-1 could be blocked by roscovitine, consistent with phosphorylation by cyclin-dependent kinases. In vitro studies with purified NHERF-1 fusion proteins and purified kinases revealed that NHERF-1 was robustly phosphorylated by the cyclin-dependent kinase Cdc2. In contrast, the NHERF-1 relative NHERF-2 was not phosphorylated at all by Cdc2. NHERF-1 possesses two serines (Ser(279) and Ser(301)) that conform to the SPX(K/R) motif preferred for phosphorylation by Cdc2. Mutation of either of these serines reduced Cdc2-mediated phosphorylation of NHERF-1 in vitro, and mutation of both residues together completely abolished Cdc2-mediated phosphorylation. When the S279A/S301A NHERF-1 mutant was expressed in cells, it failed to exhibit the mitosis phase-dependent phosphorylation observed with wild-type NHERF-1. Mutation of both Ser(279) and Ser(301) to aspartate, to mimic Cdc2 phosphorylation of NHERF-1, resulted in a NHERF-1 mutant with a markedly impaired ability to oligomerize in vitro. Similarly, endogenous NHERF-1 from lysates of mitosis phase HeLa cells exhibited a markedly reduced ability to oligomerize relative to endogenous NHERF-1 from lysates of interphase HeLa cells. Mitosis phase NHERF-1 furthermore exhibited the ability to associate with Pin1, a WW domain-containing peptidylprolyl isomerase that does not detectably bind to NHERF-1 in interphase lysates. The association of NHERF-1 with Pin1 facilitated dephosphorylation of NHERF-1, as shown in experiments in which cellular Pin1 activity was blocked by the selective inhibitor juglone. These data reveal that cellular NHERF-1 is phosphorylated during mitosis phase by Cdc2 at Ser(279) and Ser(301) and that this phosphorylation regulates NHERF-1 oligomerization and association with Pin1.

Amino Acid Sequence↗
PubMed2002

Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635.

Endothelial nitric-oxide synthase (eNOS) is regulated by signaling pathways involving multiple sites of phosphorylation. The coordinated phosphorylation of eNOS at Ser(1179) and dephosphorylation at Thr(497) activates the enzyme, whereas inhibition results when Thr(497) is phosphorylated and Ser(1179) is dephosphorylated. We have identified two further phosphorylation sites, at Ser(617) and Ser(635), by phosphopeptide mapping and matrix-assisted laser desorption ionization time of flight mass spectrometry. Purified protein kinase A (PKA) phosphorylates both sites in purified eNOS, whereas purified Akt phosphorylates only Ser(617). In bovine aortic endothelial cells, bradykinin (BK), ATP, and vascular endothelial growth factor stimulate phosphorylation of both sites. BK-stimulated phosphorylation of Ser(617) is Ca(2+)-dependent and is partially inhibited by LY294002 and wortmannin, phosphatidylinositol 3-kinase inhibitors, suggesting signaling via Akt. BK-stimulated phosphorylation of Ser(635) is Ca(2+)-independent and is completely abolished by the PKA inhibitor, KT5720, suggesting signaling via PKA. Activation of PKA with isobutylmethylxanthine also causes Ser(635), but not Ser(617), phosphorylation. Mimicking phosphorylation at Ser(635) by Ser to Asp mutation results in a greater than 2-fold increase in activity of the purified protein, whereas mimicking phosphorylation at Ser(617) does not alter maximal activity but significantly increases Ca(2+)-calmodulin sensitivity. These data show that phosphorylation of both Ser(617) and Ser(635) regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process.

Adenosine Triphosphate↗
PubMed2003

Toll-like receptors 2 and 4 activate STAT1 serine phosphorylation by distinct mechanisms in macrophages.

Engagement of Toll-like receptor (TLR) proteins activates multiple signal transduction pathways. These studies show that engagement of TLR2 and TLR4 leads to rapid phosphorylation of the transcription factor STAT1 at serine 727 (Ser-727 STAT1) in murine macrophages. Only TLR4 engagement induced STAT1 phosphorylation at tyrosine 701, although this response was delayed compared with Ser-727 STAT1 phosphorylation. Inhibition of phosphatidylinositol 3'-kinase using LY294002 blocked TLR4-induced STAT1 tyrosine phosphorylation, but this inhibitor had no effect on STAT1 serine phosphorylation. TLR-induced phosphorylation of Ser-727 STAT1 could be blocked by the selective p38 mitogen-activated protein kinase inhibitor SB203580. However, activation of p38 was not sufficient to induce Ser-727 STAT1 phosphorylation in macrophages. TLR2-induced activation of Ser-727 STAT1 phosphorylation required the adapter protein MyD88, whereas TLR4-induced activation of Ser-727 STAT1 phosphorylation was not solely dependent on MyD88. Lastly, TLR4-induced activation of Ser-727 STAT1 phosphorylation could be blocked by rottlerin, a specific inhibitor of protein kinase C-delta. In contrast, rottlerin had no effect on STAT1 phosphorylation induced via TLR2. Together, these data demonstrate that activation STAT1 tyrosine and serine phosphorylation are distinct consequences of TLR engagement in murine macrophages. Furthermore, p38 mitogen-activated protein kinase, protein kinase C-delta, and a novel TLR2-specific signaling pathway appear to be necessary to induce Ser-727 STAT1 phosphorylation.

Animals↗
PubMed2004

In vitro phosphorylation by cAMP-dependent protein kinase up-regulates recombinant Saccharomyces cerevisiae mannosylphosphodolichol synthase.

DPM1 is the structural gene for mannosylphosphodolichol synthase (i.e. Dol-P-Man synthase, DPMS) in Saccharomyces cerevisiae. Earlier studies with cDNA cloning and sequence analysis have established that 31-kDa DPMS of S. cerevisiae contains a consensus sequence (YRRVIS141) that can be phosphorylated by cAMP-dependent protein kinase (PKA). We have been studying the up-regulation of DPMS activity by protein kinase A-mediated phosphorylation in higher eukaryotes, and used the recombinant DPMS from S. cerevisiae in this study to advance our knowledge further. DPMS catalytic activity was indeed enhanced severalfold when the recombinant protein was phosphorylated in vitro. The rate as well as the magnitude of catalysis was higher with the phosphorylated enzyme. A similar increase in the catalytic activity was also observed when the in vitro phosphorylated recombinant DPMS was assayed as a function of increasing concentrations of exogenous dolichylmonophosphate (Dol-P). Kinetic studies indicated that there was no change in the Km for GDP-mannose between the in vitro phosphorylated and control recombinant DPMS, but the Vmax was increased by 6-fold with the phosphorylated enzyme. In vitro phosphorylated recombinant DPMS also exhibited higher enzyme turnover (kcat) and enzyme efficiency (kcat/Km). SDS-PAGE followed by autoradiography of the 32P-labeled DPMS detected a 31-kDa phosphoprotein, and immunoblotting with anti-phosphoserine antibody established the presence of a phosphoserine residue in in vitro phosphorylated recombinant DPMS. To confirm the phosphorylation activation of recombinant DPMS, serine 141 in the consensus sequence was replaced with alanine by PCR site-directed mutagenesis. The S141A DPMS mutant exhibited more than half-a-fold reduction in catalytic activity compared with the wild type when both were analyzed after in vitro phosphorylation. Thus, confirming that S. cerevisiae DPMS activity is indeed regulated by the cAMP-dependent protein phosphorylation signal, and the phosphorylation target is serine 141.

Catalysis↗
PubMed2004

Protein kinase C mediates phosphorylation, desensitization, and trafficking of the D2 dopamine receptor.

Previously, D2 dopamine receptors (D2 DARs) have been shown to undergo G-protein-coupled receptor kinase phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second messenger-activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC), to mediate phosphorylation and desensitization of the D2 DAR. HEK293T cells were transiently transfected with the D2 DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2 DAR, suggesting that PKA does not phosphorylate the D2 DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in an approximately 3-fold increase in D2 DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by phorbol 12,13-dibutyrate, but not by 4alpha-phorbol 12,13-didecanoate, active and inactive, phorbol diesters, respectively. The PMA-mediated D2 DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II, and augmented by co-transfection with PKCbetaI. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation, suggesting that PKC is not involved in this response. PKC phosphorylation of the D2 DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Most interestingly, PKC phosphorylation also promoted internalization of the D2 DAR through a beta-arrestin- and dynamin-dependent pathway, a response not usually associated with PKC phosphorylation of G-protein-coupled receptors. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the third intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D2 DAR, whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D2 DAR, resulting in both functional desensitization and receptor internalization.

Amino Acid Sequence↗
PubMed2005

Protein kinase C phosphorylation of the metabotropic glutamate receptor mGluR5 on Serine 839 regulates Ca2+ oscillations.

The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1alpha, triggers intracellular calcium release; however, mGluR5 activation is unique in that it elicits Ca2+ oscillations. A short region of the mGluR5 C terminus is the critical determinant and differs from the analogous region of mGluR1alpha by a single amino acid residue, Thr-840, which is an aspartic acid (Asp-854) in mGluR1alpha. Previous studies show that mGluR5-elicited Ca2+ oscillations require protein kinase C (PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylation site. However, direct phosphorylation of mGluR5 has not been studied in detail. We have used biochemical analyses to directly investigate the phosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 is directly phosphorylated by PKC, whereas Thr-840 plays a permissive role. Although Ser-839 is conserved in mGluR1alpha (Ser-853), it is not phosphorylated, as the adjacent residue (Asp-854) is not permissive; however, mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylation of Ser-853 on mGluR1alpha. We investigated the physiological consequences of mGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations that eliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependent Ca2+ oscillations. However, mutation of Thr-840 to alanine, which prevents potential Thr-840 phosphorylation but is still permissive for Ser-839 phosphorylation, has no effect on Ca2+ oscillations. Thus, we showed that it is phosphorylation of Ser-839, not Thr-840, that is absolutely required for the unique Ca2+ oscillations produced by mGluR5 activation. The Thr-840 residue is important only in that it is permissive for the PKC-dependent phosphorylation of Ser-839.

Amino Acid Sequence↗
PubMed2005

The phosphorylation of Ser318 of insulin receptor substrate 1 is not per se inhibitory in skeletal muscle cells but is necessary to trigger the attenuation of the insulin-stimulated signal.

The Ser/Thr phosphorylation of insulin receptor substrate 1 (IRS) is one key mechanism to stimulate and/or attenuate insulin signal transduction. Using a phospho-specific polyclonal antibody directed against phosphorylated Ser(318) of IRS-1, we found a rapid and transient insulin-stimulated phosphorylation of Ser(318) in human and rodent skeletal muscle cell models and in muscle tissue of insulin-treated mice. None of the investigated insulin resistance-associated factors (e.g. high glucose, tumor necrosis factor-alpha, adrenaline) stimulated the phosphorylation of Ser(318). Studying the function of this phosphorylation, we found that replacing Ser(318) by alanine completely prevented both the attenuation of insulin-stimulated Akt/protein kinase B Ser(473) phosphorylation and glucose uptake after 60 min of insulin stimulation. Unexpectedly, after acute insulin stimulation, we observed that phosphorylation of Ser(318) is not inhibitory but rather enhances insulin signal transduction because introduction of Ala(318) led to a reduction of the insulin-stimulated Akt/protein kinase B phosphorylation. Furthermore, replacing Ser(318) by glutamate, i.e. mimicking phosphorylation, improved glucose uptake after acute insulin stimulation. These data suggest that phosphorylation of Ser(318) is not per se inhibitory but is necessary to trigger the attenuation of the insulin-stimulated signal in skeletal muscle cells. Investigating the molecular mechanism of insulin-stimulated Ser(318) phosphorylation, we found that phosphatidylinositol 3-kinase-mediated activation of atypical protein kinase C-zeta and recruitment of protein kinase C-zeta to IRS-1 was responsible for this phosphorylation. We conclude that Ser(318) phosphorylation of IRS-1 is an early physiological event in insulin-stimulated signal transduction, which attenuates the continuing action of insulin.

Amino Acid Sequence↗
PubMed2005

DNA replication stress-induced phosphorylation of cyclic AMP response element-binding protein mediated by ATM.

The DNA damage-response regulators ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) are structurally and functionally related protein kinases that exhibit nearly identical substrate specificities in vitro. Current paradigms hold that the relative contributions of ATM and ATR to nuclear substrate phosphorylation are dictated by the type of initiating DNA lesion; ATM-dependent substrate phosphorylation is principally activated by DNA double strand breaks, whereas ATR-dependent substrate phosphorylation is induced by UV light and other forms of DNA replication stress. In this report, we employed the cyclic AMP-response element-binding (CREB) protein to provide evidence for substrate discrimination by ATM and ATR in cellulo. ATM and ATR phosphorylate CREB in vitro, and CREB is phosphorylated on Ser-121 in intact cells in response to ionizing radiation (IR), UV light, and hydroxyurea. The UV light- and hydroxyurea-induced phosphorylation of CREB was delayed in comparison to the canonical ATR substrate CHK1, suggesting potentially different mechanisms of phosphorylation. UV light-induced CREB phosphorylation temporally correlated with ATM autophosphorylation on Ser-1981, and an ATM-specific small interfering RNA suppressed CREB phosphorylation in response to this stimulus. UV light-induced CREB phosphorylation was absent in ATM-deficient cells, confirming that ATM is required for CREB phosphorylation in UV irradiation-damaged cells. Interestingly, RNA interference-mediated suppression of ATR partially inhibited CREB phosphorylation in response to UV light, which correlated with reduced phosphorylation of ATM on Ser-1981. These findings suggest that ATM is the major genotoxin-induced CREB kinase in mammalian cells and that ATR lies upstream of ATM in a UV light-induced signaling pathway.

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