scholarly journals Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10

1998 ◽  
Vol 18 (6) ◽  
pp. 3289-3299 ◽  
Author(s):  
Dongqing Huang ◽  
Jason Moffat ◽  
Wayne A. Wilson ◽  
Lynda Moore ◽  
Christine Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acid Phosphatase ◽  
Protein Kinase ◽  
Substrate Specificity ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glycogen Accumulation ◽  
Acid Phosphatase Gene

ABSTRACT In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlikepho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation ofPHO85 suppressed the glycogen storage deficiency ofsnf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

scholarly journals Overexpression of SH2-Containing Inositol Phosphatase 2 Results in Negative Regulation of Insulin-Induced Metabolic Actions in 3T3-L1 Adipocytes via Its 5′-Phosphatase Catalytic Activity

2001 ◽  
Vol 21 (5) ◽  
pp. 1633-1646 ◽  
Author(s):  
Tsutomu Wada ◽  
Toshiyasu Sasaoka ◽  
Makoto Funaki ◽  
Hiroyuki Hori ◽  
Shihou Murakami ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Phosphatase Activity ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Dominant Negative ◽  
Inositol Phosphatase

ABSTRACT Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5′-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5′-phosphatase-defective SHIP2 (ΔIP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor β subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or ΔIP-SHIP2. Because WT-SHIP2 possesses the 5′-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of ΔIP-SHIP2, indicating that ΔIP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase Cλ in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase Cλ, whereas these activations were increased by expression of ΔIP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of ΔIP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3β and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of ΔIP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5′-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.

scholarly journals Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (8) ◽  
pp. 4357-4365 ◽  
Author(s):  
D Huang ◽  
I Farkas ◽  
P J Roach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Mutant Gene ◽  
Glycogen Synthase Kinase ◽  
Partial Purification ◽  
Glycogen Accumulation ◽  
Dependent Protein

In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.

scholarly journals c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in αT3-1 Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 839-849 ◽  
Author(s):  
Buffy S. Ellsworth ◽  
Brett R. White ◽  
Ann T. Burns ◽  
Brian D. Cherrington ◽  
Annette M. Otis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cell Model ◽  
Receptor Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Activator Protein

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in αT3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH3). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in αT3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.

scholarly journals Antagonistic Controls of Autophagy and Glycogen Accumulation by Snf1p, the Yeast Homolog of AMP-Activated Protein Kinase, and the Cyclin-Dependent Kinase Pho85p

2001 ◽  
Vol 21 (17) ◽  
pp. 5742-5752 ◽  
Author(s):  
Zhong Wang ◽  
Wayne A. Wilson ◽  
Marie A. Fujino ◽  
Peter J. Roach
Keyword(s):  
Protein Kinase ◽  
Stationary Phase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glycogen Storage ◽  
Wild Type ◽  
Gene Encoding ◽  
Glycogen Accumulation ◽  
Yeast Homolog ◽  
Glycogen Stores

ABSTRACT In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of otherSNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in ansnf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1control of glycogen synthesis. Induction of autophagy inpho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.

scholarly journals Mutations in the Gal83 Glycogen-Binding Domain Activate the Snf1/Gal83 Kinase Pathway by a Glycogen-Independent Mechanism

2004 ◽  
Vol 24 (1) ◽  
pp. 352-361 ◽  
Author(s):  
Heather A. Wiatrowski ◽  
Bryce J. W. van Denderen ◽  
Cristin D. Berkey ◽  
Bruce E. Kemp ◽  
David Stapleton ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Metabolic Stress ◽  
Binding Domain ◽  
Glycogen Accumulation ◽  
Snf1 Kinase ◽  
Transcriptional Regulatory ◽  
Amp Activated Protein Kinase

ABSTRACT The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK β1 subunit, and the sequence is conserved in the Snf1 kinase β subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK β1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

scholarly journals BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

2006 ◽  
Vol 401 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Gopal P. Sapkota ◽  
Lorna Cummings ◽  
Felicity S. Newell ◽  
Christopher Armstrong ◽  
Jennifer Bain ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Phorbol Ester ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Camp Response Element Binding ◽  
S6 Kinase ◽  
Ribosomal S6 Kinase

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC50 of 10–30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3β and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.

scholarly journals Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts.

1992 ◽  
Vol 12 (10) ◽  
pp. 4694-4705 ◽  
Author(s):  
S J Baker ◽  
T K Kerppola ◽  
D Luk ◽  
M T Vandenberg ◽  
D R Marshak ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Glycogen Synthase ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Serum Stimulation ◽  
Dependent Protein

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.

scholarly journals Pioglitazone Protected against Cardiac Hypertrophy via Inhibiting AKT/GSK3βand MAPK Signaling Pathways

PPAR Research ◽  
2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Wen-Ying Wei ◽  
Zhen-Guo Ma ◽  
Si-Chi Xu ◽  
Ning Zhang ◽  
Qi-Zhu Tang
Keyword(s):  
Protein Kinase ◽  
Cardiac Hypertrophy ◽  
Glycogen Synthase ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Neonatal Rat ◽  
Aortic Banding ◽  
Hypertrophic Response

Peroxisome proliferator activated receptorγ(PPARγ) has been closely involved in the process of cardiovascular diseases. This study was to investigate whether pioglitazone (PIO), a PPARγagonist, could protect against pressure overload-induced cardiac hypertrophy. Mice were orally given PIO (2.5 mg/kg) from 1 week after aortic banding and continuing for 7 weeks. The morphological examination and biochemical analysis were used to evaluate the effects of PIO. Neonatal rat ventricular cardiomyocytes were also used to verify the protection of PIO against hypertrophy in vitro. The results in our study demonstrated that PIO remarkably inhibited hypertrophic response induced by aortic banding in vivo. Besides, PIO also suppressed cardiac fibrosis in vivo. PIO treatment also inhibited the activation of protein kinase B (AKT)/glycogen synthase kinase-3β(GSK3β) and mitogen-activated protein kinase (MAPK) in the heart. In addition, PIO alleviated angiotensin II-induced hypertrophic response in vitro. In conclusion, PIO could inhibit cardiac hypertrophy via attenuation of AKT/GSK3βand MAPK pathways.

scholarly journals Amylin impairment of insulin effects on glycogen synthesis and phosphoenolpyruvate carboxykinase gene expression in rat primary cultured hepatocytes

1994 ◽  
Vol 304 (2) ◽  
pp. 449-453 ◽  
Author(s):  
S Baqué ◽  
J J Guinovart ◽  
A M Gómez-Foix
Keyword(s):  
Gene Expression ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Mrna Level ◽  
Rat Hepatocytes ◽  
Synthesis Rate ◽  
Cultured Hepatocytes ◽  
Glycogen Accumulation ◽  
Primary Cultured Hepatocytes

The ability of amylin to impair hepatic insulin action is controversial. We have found that the effect of amylin in primary cultured hepatocytes is strongly dependent on the culture conditions. Only in hepatocytes preincubated in the presence of fetal serum did amylin, at concentrations ranging from 1 to 100 nM, reduce insulin-stimulated glycogen synthesis rate and glycogen accumulation without showing direct effects. Neither basal glycogen synthase nor glycogen phosphorylase activity was modified by amylin treatment. Nevertheless, amylin (100 nM) blocked the activation of glycogen synthase by insulin. Amylin also proved capable of opposing the reduction in the expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene induced by insulin, whereas the basal mRNA level of PEPCK was unaffected by amylin treatment. Thus, these results show that, in cultured rat hepatocytes, amylin is indeed able to interfere with insulin regulation of glycogenesis and PEPCK gene expression, favouring the hypothesis that amylin may modulate liver sensitivity to insulin.

scholarly journals Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

2004 ◽  
Vol 24 (16) ◽  
pp. 7298-7311 ◽  
Author(s):  
Keith Wheaton ◽  
Karl Riabowol
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Serum Response Factor ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Response Factor ◽  
Serum Response

ABSTRACT Fibroblasts lose the ability to replicate in response to growth factors and become unable to express growth-associated immediate-early genes, including c-fos and egr-1, as they become senescent. The serum response factor (SRF), a major transcriptional activator of immediate-early gene promoters, loses the ability to bind to the serum response element (SRE) and becomes hyperphosphorylated in senescent cells. We identify protein kinase C delta (PKCδ) as the kinase responsible for inactivation of SRF both in vitro and endogenously in senescent cells. This is due to a higher level of PKCδ activity as cells age, production of the PKCδ catalytic fragment, and its nuclear localization in senescent but not in low-passage-number cells. The phosphorylation of T160 of SRF by PKCδ in vitro and in vivo led to loss of SRF DNA binding activity. Both the PKCδ inhibitor rottlerin and ectopic expression of a dominant negative form of PKCδ independently restored SRE-dependent transcription and immediate-early gene expression in senescent cells. Modulation of PKCδ activity in vivo with rottlerin or bistratene A altered senescent- and young-cell morphology, respectively. These observations support the idea that the coordinate transcriptional inhibition of several growth-associated genes by PKCδ contributes to the senescent phenotype.

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