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Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity.
Kumar A, Harris TE, Keller SR, Choi KM, Magnuson MA, Lawrence JC
(2008) Mol Cell Biol 28: 61-70
MeSH Terms: Animals, Biological Transport, Carrier Proteins, GTPase-Activating Proteins, Gene Expression Regulation, Glucose, Glycogen, Glycogen Synthase, Insulin, Male, Mice, Mice, Knockout, Muscles, Phosphorylation, Phosphoserine, Phosphothreonine, Proto-Oncogene Proteins c-akt, Rapamycin-Insensitive Companion of mTOR Protein
Show Abstract · Added August 22, 2011
Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.
3 Communities
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18 MeSH Terms
Global assessment of regulation of phosphorylation of insulin receptor substrate-1 by insulin in vivo in human muscle.
Yi Z, Langlais P, De Filippis EA, Luo M, Flynn CR, Schroeder S, Weintraub ST, Mapes R, Mandarino LJ
(2007) Diabetes 56: 1508-16
MeSH Terms: Adult, Biopsy, Blood Glucose, Female, Glucose Clamp Technique, Glucose Tolerance Test, Homeostasis, Humans, Insulin, Insulin Receptor Substrate Proteins, Kinetics, Male, Muscle, Skeletal, Phosphoproteins, Phosphorylation, Phosphoserine, Phosphothreonine, Reference Values
Show Abstract · Added December 10, 2013
OBJECTIVE - Research has focused on insulin receptor substrate (IRS)-1 as a locus for insulin resistance. Tyrosine phosphorylation of IRS-1 initiates insulin signaling, whereas serine/threonine phosphorylation alters the ability of IRS-1 to transduce the insulin signal. Of 1,242 amino acids in IRS-1, 242 are serine/threonine. Serine/threonine phosphorylation of IRS-1 is affected by many factors, including insulin. The purpose of this study was to perform global assessment of phosphorylation of serine/threonine residues in IRS-1 in vivo in humans.
RESEARCH DESIGN AND METHODS - In this study, we describe our use of capillary high-performance liquid chromotography electrospray tandem mass spectrometry to identify/quantify site-specific phosphorylation of IRS-1 in human vastus lateralis muscle obtained by needle biopsy basally and after insulin infusion in four healthy volunteers.
RESULTS - Twenty-two serine/threonine phosphorylation sites were identified; 15 were quantified. Three sites had not been previously identified (Thr495, Ser527, and S1005). Insulin increased the phosphorylation of Ser312, Ser616, Ser636, Ser892, Ser1101, and Ser1223 (2.6 +/- 0.4, 2.9 +/- 0.8, 2.1 +/- 0.3, 1.6 +/- 0.1, 1.3 +/- 0.1, and 1.3 +/- 0.1-fold, respectively, compared with basal; P < 0.05); phosphorylation of Ser348, Thr446, Thr495, and Ser1005 decreased (0.4 +/- 0.1, 0.2 +/- 0.1, 0.1 +/- 0.1, and 0.3 +/- 0.2-fold, respectively; P < 0.05).
CONCLUSIONS - These results provide an assessment of IRS-1 phosphorylation in vivo and show that insulin has profound effects on IRS-1 serine/threonine phosphorylation in healthy humans.
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18 MeSH Terms
Temporal dissociation of frequency-dependent acceleration of relaxation and protein phosphorylation by CaMKII.
Huke S, Bers DM
(2007) J Mol Cell Cardiol 42: 590-9
MeSH Terms: Animals, Calcium-Binding Proteins, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium-Calmodulin-Dependent Protein Kinases, Enzyme Inhibitors, Heart, Muscle Cells, Phosphoric Monoester Hydrolases, Phosphorylation, Phosphoserine, Phosphothreonine, Rats, Ryanodine Receptor Calcium Release Channel, Time Factors
Show Abstract · Added May 27, 2014
Frequency-dependent acceleration of relaxation (FDAR) is an important intrinsic mechanism that allows for diastolic filling of the ventricle at higher heart rates, yet its molecular mechanism is still not understood. Previous studies showed that FDAR is dependent on functional sarcoplasmic reticulum (SR) and can be abolished by phosphatase or by Ca/CaM kinase (CaMKII) inhibition. Additionally, CaMKII activity/autophosphorylation has been shown to be frequency-dependent. Thus, we tested the hypothesis that CaMKII phosphorylation of SR Ca(2+)-handling proteins (Phospholamban (PLB), Ca(2+) release channel (RyR)) mediates FDAR. Here we show that FDAR occurs abruptly in fluo-4 loaded isolated rat ventricular myocytes when frequency is raised from 0.1 to 2 Hz. The effect is essentially complete within four beats (2 s) with the tau of [Ca(2+)](i) decline decreasing by 42+/-3%. While there is a detectable increase in PLB Thr-17 and RyR Ser-2814 phosphorylation, the increase is quantitatively small (PLB<5%, RyR approximately 8%) and the time-course is clearly delayed with regard to FDAR. The low substrate phosphorylation indicates that pacing of myocytes only mildly activates CaMKII and consistent with this CaMKIIdelta autophosphorylation did not increase with pacing alone. However, in the presence of phosphatase 1 inhibition pacing triggered a net-increase in autophosphorylated CaMKII and also greatly enhanced PLB and RyR phosphorylation. We conclude that FDAR does not rely on phosphorylation of PLB or RyR. Even though CaMKII does become activated when myocytes are paced, phosphatases immediately antagonize CaMKII action, limit substrate phosphorylation and also prevent sustained CaMKII autophosphorylation (thereby suppressing global CaMKII effects).
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14 MeSH Terms
L-type Ca2+ channel facilitation mediated by phosphorylation of the beta subunit by CaMKII.
Grueter CE, Abiria SA, Dzhura I, Wu Y, Ham AJ, Mohler PJ, Anderson ME, Colbran RJ
(2006) Mol Cell 23: 641-50
MeSH Terms: Amino Acid Sequence, Animals, Calcium Channels, L-Type, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium-Calmodulin-Dependent Protein Kinases, Cells, Cultured, Humans, Ion Channel Gating, Mice, Molecular Sequence Data, Myocytes, Cardiac, Phosphorylation, Phosphothreonine, Protein Binding, Protein Subunits, Rats, Rats, Sprague-Dawley, Recombinant Proteins
Show Abstract · Added June 21, 2013
L-type Ca(2+) channels (LTCCs) are major entry points for Ca(2+) in many cells. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is associated with cardiac LTCC complexes and increases channel open probability (P(O)) to dynamically increase Ca(2+) current (I(Ca)) and augment cellular Ca(2+) signaling by a process called facilitation. However, the critical molecular mechanisms for CaMKII localization to LTCCs and I(Ca) facilitation in cardiomyocytes have not been defined. We show CaMKII binds to the LTCC beta(2a) subunit and preferentially phosphorylates Thr498 in beta(2a). Mutation of Thr498 to Ala (T498A) in beta(2a) prevents CaMKII-mediated increases in the P(O) of recombinant LTCCs. Moreover, expression of beta(2a)(T498A) in adult cardiomyocytes ablates CaMKII-mediated I(Ca) facilitation, demonstrating that phosphorylation of beta(2a) at Thr498 modulates native calcium channels. These findings reveal a molecular mechanism for targeting CaMKII to LTCCs and facilitating I(Ca) that may modulate Ca(2+) entry in diverse cell types coexpressing CaMKII and the beta(2a) subunit.
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18 MeSH Terms
Phosphorylation of p27Kip1 at Thr-157 interferes with its association with importin alpha during G1 and prevents nuclear re-entry.
Shin I, Rotty J, Wu FY, Arteaga CL
(2005) J Biol Chem 280: 6055-63
MeSH Terms: Active Transport, Cell Nucleus, Animals, Cell Cycle Proteins, Cell Line, Cell Nucleus, Cyclin-Dependent Kinase Inhibitor p27, Cytosol, G1 Phase, Humans, Mice, Models, Biological, Phosphorylation, Phosphoserine, Phosphothreonine, Protein Binding, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, Time Factors, Tumor Suppressor Proteins, alpha Karyopherins
Show Abstract · Added March 5, 2014
We have studied mechanisms of Akt-mediated phosphorylation and regulation of cellular localization of p27. Akt phosphorylates Thr-157 in p27 and retains it in the cytosol. In cells arrested in G(1) and then synchronized to enter into S phase, Akt-mediated phosphorylation of Thr-157 p27 occurred in the cytosol during G(1) phase of the cell cycle. Both T157A and S10A p27 mutants localized in the nucleus in all phases of the cell cycle regardless of the expression of active Akt. Thr-157 phosphorylation was undetectable in S10A-p27, suggesting that Ser-10 phosphorylation is required for p27 localization in the cytosol and subsequent phosphorylation at Thr-157. Phosphorylation at Thr-157 interrupted the association of p27 with importin alpha. A T157A-p27 mutant protein exhibited higher association with importin alpha than wild-type-p27. Treatment of transfected and endogenous p27 with alkaline phosphatase rescued its association with importin alpha. Leptomycin B inhibited cytosolic Thr-157 P-p27 staining, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation. Heterokaryon shuttling assays with NIH3T3 (mouse) cells transfected with FLAG-p27 and HeLa (human) cells revealed that both wild type and T157A-p27 shuttled from NIH3T3 to HeLa cell nuclei with similar frequencies. However, S10A-p27 was found only in the NIH3T3 nuclei of NIH3T3-HeLa cell fusions. These results suggest that 1) Ser-10 phosphorylation is required for nuclear export of p27, 2) subsequent Akt-mediated phosphorylation at Thr-157 during G(1) phase corrals p27 in the cytosol, and 3) Thr-157 phosphorylation inhibits the association of p27 with importin alpha thus preventing its re-entry into the nucleus.
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21 MeSH Terms
Threonine 180 is required for G-protein-coupled receptor kinase 3- and beta-arrestin 2-mediated desensitization of the mu-opioid receptor in Xenopus oocytes.
Celver JP, Lowe J, Kovoor A, Gurevich VV, Chavkin C
(2001) J Biol Chem 276: 4894-900
MeSH Terms: Animals, Arrestins, Dose-Response Relationship, Drug, Down-Regulation, Enkephalin, Ala(2)-MePhe(4)-Gly(5)-, G Protein-Coupled Inwardly-Rectifying Potassium Channels, G-Protein-Coupled Receptor Kinase 3, Mutagenesis, Site-Directed, Oocytes, Phosphothreonine, Potassium Channels, Potassium Channels, Inwardly Rectifying, Protein-Serine-Threonine Kinases, Receptors, Opioid, mu, Transfection, Xenopus, beta-Arrestins
Show Abstract · Added December 10, 2013
To determine the sites in the mu-opioid receptor (MOR) critical for agonist-dependent desensitization, we constructed and coexpressed MORs lacking potential phosphorylation sites along with G-protein activated inwardly rectifying potassium channels composed of K(ir)3.1 and K(ir)3.4 subunits in Xenopus oocytes. Activation of MOR by the stable enkephalin analogue, [d-Ala(2),MePhe(4),Glyol(5)]enkephalin, led to homologous MOR desensitization in oocytes coexpressing both G-protein-coupled receptor kinase 3 (GRK3) and beta-arrestin 2 (arr3). Coexpression with either GRK3 or arr3 individually did not significantly enhance desensitization of responses evoked by wild type MOR activation. Mutation of serine or threonine residues to alanines in the putative third cytoplasmic loop and truncation of the C-terminal tail did not block GRK/arr3-mediated desensitization of MOR. Instead, alanine substitution of a single threonine in the second cytoplasmic loop to produce MOR(T180A) was sufficient to block homologous desensitization. The insensitivity of MOR(T180A) might have resulted either from a block of arrestin activation or arrestin binding to MOR. To distinguish between these alternatives, we expressed a dominant positive arrestin, arr2(R169E), that desensitizes G protein-coupled receptors in an agonist-dependent but phosphorylation-independent manner. arr2(R169E) produced robust desensitization of MOR and MOR(T180A) in the absence of GRK3 coexpression. These results demonstrate that the T180A mutation probably blocks GRK3- and arr3-mediated desensitization of MOR by preventing a critical agonist-dependent receptor phosphorylation and suggest a novel GRK3 site of regulation not yet described for other G-protein-coupled receptors.
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17 MeSH Terms
Overexpression of c-Myc and cell immortalization alters c-Myc phosphorylation.
Lutterbach B, Hann SR
(1997) Oncogene 14: 967-75
MeSH Terms: 3T3 Cells, Amino Acid Sequence, Animals, Cell Survival, Cell Transformation, Neoplastic, Mice, Molecular Sequence Data, Peptide Mapping, Phosphorylation, Phosphoserine, Phosphothreonine, Proto-Oncogene Proteins c-myc, Sequence Deletion, Structure-Activity Relationship, Transcriptional Activation, Tumor Cells, Cultured
Show Abstract · Added March 5, 2014
Using an extensive series of deletion and site-specific mutation constructs, we have identified five new phosphorylation sites in c-Myc in the N-terminal transactivation domain and near the C-terminal DNA binding/heterodimerization domain. We have also found that Thr-58 phosphorylation is regulated by specific cellular events. When c-Myc is overexpressed in cells Thr-58 phosphorylation was greatly enhanced in the overexpressed, exogenous c-Myc as compared with the endogenous protein. In contrast, an inhibition of Thr-58 phosphorylation and an enhancement of Serine 62 phosphorylation was observed in c-Myc from immortalized cells compared with primary cells. No significant changes in c-Myc phosphorylation were found when transformed and nontransformed cells were compared. Finally, mutations at these phosphorylation sites, either individually or in combination with previously described sites, did not affect the ability of c-Myc to transactivate through the CACGTG Myc/Max DNA binding sites. These results further suggest that either the molecular role for c-Myc phosphorylation does not involve modulating transcriptional activity of c-Myc or that the CACGTG site does not represent a physiological promoter element.
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16 MeSH Terms
Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.
Lutterbach B, Hann SR
(1994) Mol Cell Biol 14: 5510-22
MeSH Terms: Amino Acid Sequence, Animals, Cell Line, Cell Nucleus, Chick Embryo, Coturnix, Cytoplasm, Mitogens, Mitosis, Molecular Sequence Data, Phosphorylation, Phosphoserine, Phosphothreonine, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins c-myc, Transcriptional Activation
Show Abstract · Added March 5, 2014
The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.
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16 MeSH Terms
The protein-tyrosine kinase substrate, p81, is homologous to a chicken microvillar core protein.
Gould KL, Cooper JA, Bretscher A, Hunter T
(1986) J Cell Biol 102: 660-9
MeSH Terms: Animals, Carrier Proteins, Cell Line, Cytoskeletal Proteins, Fluorescent Antibody Technique, Humans, Isoelectric Point, Microfilament Proteins, Microvilli, Molecular Weight, Peptide Fragments, Phosphoproteins, Phosphoserine, Phosphothreonine, Phosphotyrosine, Protein-Tyrosine Kinases, Subcellular Fractions, Tissue Distribution, Tyrosine
Show Abstract · Added March 5, 2014
p81, a protein-tyrosine kinase substrate previously identified in epidermal growth factor-treated A431 cells, is demonstrated to be homologous to ezrin, an 80-kD component of microvillar core proteins. p81 has been characterized using antiserum raised against purified chicken intestinal ezrin. p81, located by indirect immunofluorescent staining, is concentrated in surface projections of A431 cells such as microvilli and retraction fibers. None of the conditions of biochemical cell fractionation tested completely solubilizes p81; the insoluble p81 partitions as if associated with the cytoskeleton. The soluble form of p81 behaves as a monomer in all extraction procedures studied. EGF-stimulated phosphorylation of p81 does not appear to change its intracellular location. p81 exhibits a wide tissue distribution with highest levels of expression in small intestine, kidney, thymus, and lung. Intermediate levels are found in spleen, thymus, lymph nodes, and bone marrow, with low levels in brain, heart, and testes. p81 is undetectable in muscle and liver. In A431 cells, p81 is phosphorylated on serine and threonine residues. Upon EGF treatment, approximately 10% of p81 becomes phosphorylated on tyrosine, and the phosphorylation of threonine residues increases.
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19 MeSH Terms
Casein kinase I phosphorylates the 25-kDa mRNA cap-binding protein.
Haas DW, Hagedorn CH
(1991) Arch Biochem Biophys 284: 84-9
MeSH Terms: Animals, Casein Kinases, Eukaryotic Initiation Factor-4E, Humans, In Vitro Techniques, Peptide Initiation Factors, Phosphorylation, Phosphoserine, Phosphothreonine, Protein Kinases, RNA Caps, Rabbits
Show Abstract · Added March 13, 2015
The 25-kDa mRNA cap-binding protein (eIF-4E) exists in both phosphorylated and dephosphorylated forms in eukaryotic cells. Phosphorylated eIF-4E appears to be preferentially associated with 48 S initiation complexes and with the 220-kDa subunit of eIF-4F. In addition, dephosphorylation of eIF-4E has been observed during heat shock and mitosis which are accompanied by decreased protein synthesis. However, the control of eIF-4E phosphorylation and its regulatory role remain poorly understood. Using eIF-4E as a substrate we have identified and purified from rabbit reticulocytes a protein kinase that phosphorylates eIF-4E in vitro. This enzyme phosphorylated eIF-4E on both serine and threonine residues with an apparent Km of 3.7 microM. The molecular mass of the enzyme and specificity for substrates other than eIF-4E suggested that this enzyme was a species of casein kinase I. This was confirmed by comparing the phosphopeptide map of the purified reticulocyte enzyme with that of rabbit skeletal muscle casein kinase I and by comparing phosphopeptide maps of eIF-4E phosphorylated in vitro by each enzyme. We conclude that casein kinase I phosphorylates eIF-4E in vitro and suggest that eIF-4E may be phosphorylated by casein kinase I in intact cells under some physiologic conditions.
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12 MeSH Terms