scholarly journals ATP citrate-lyase and glycogen synthase kinase-3β in 3T3-L1 cells during differentiation into adipocytes

1994 ◽  
Vol 300 (2) ◽  
pp. 477-482 ◽  
Author(s):  
W B Benjamin ◽  
S N Pentyala ◽  
J R Woodgett ◽  
Y Hod ◽  
D Marshak
Keyword(s):  
Glycogen Synthase ◽  
Protein Band ◽  
Glycogen Synthase Kinase ◽  
Fat Cells ◽  
Apparent Mass ◽  
Atp Citrate Lyase ◽  
Protein Levels ◽  
Citrate Lyase ◽  
Sds Page ◽  
Dependent Protein

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synthase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3T3-L1 cells during differentiation from fibroblasts into fat-cells. Protein levels were estimated from immunoblots using specific antisera. Cytosol from confluent cells contain significant amounts of GSK-3 beta, which fell during differentiation of these cells into adipocytes. CL from confluent cells was found to be mostly in the form of a single protein band of apparent mass 110 kDa. Levels of CL and ACC increased during cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a complex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phenotype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alkaline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a higher-order phosphorylation state of the same protein. Furthermore, when purified CL, mostly expressed as the 110 kDa species, was phosphorylated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated more slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was hormone-regulated, since, in samples from fat-cells that had the complex two-band pattern, when cultured in medium without serum or hormones, CL migration reverted to a single band of 110 kDa, similar to confluent cells. Treatment of these ‘down-regulated’ cells with insulin rapidly induced substantial amounts of the 113 kDa species, with a concomitant decrease in the 110 kDa species.

scholarly journals Identification of multifunctional ATP-citrate lyase kinase as the α-isoform of glycogen synthase kinase-3

1992 ◽  
Vol 288 (1) ◽  
pp. 309-314 ◽  
Author(s):  
K Hughes ◽  
S Ramakrishna ◽  
W B Benjamin ◽  
J R Woodgett
Keyword(s):  
Drosophila Melanogaster ◽  
Protein Kinase ◽  
Molecular Cloning ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Atp Citrate Lyase ◽  
Multifunctional Protein ◽  
Citrate Lyase ◽  
Biochemical Analyses

Multifunctional ATP-citrate lyase kinase (ACLK) exhibits several properties that are similar to glycogen-synthase kinase-3 (GSK-3). The molecular cloning of two distinct mammalian GSK-3 cDNAs and a Drosophila melanogaster (fruitfly) homologue, zeste-white3sgg, has established the existence of a GSK-3 subfamily. A multifunctional protein kinase first identified as an ACLK has recently been shown to exhibit several similarities to the alpha- and beta-forms of GSK-3. Here we have used immunological and biochemical analyses to directly compare these enzymes. Thus purified preparations of ACLK isolated from brain and liver preferentially cross-react with anti-GSK-3 alpha antisera and phosphorylate previously defined substrates of GSK-3 at identical sites. Conversely, both alpha- and beta-forms of GSK-3 phosphorylated ATP-citrate lyase at the same site(s) targeted by ACLK. These, and other similarities, demonstrate ACLK to be identical with, or highly related to, GSK-3 alpha, the implications of which are discussed.

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 Glycogen synthase kinase 3β mediates high glucose-induced ubiquitination and proteasome degradation of insulin receptor substrate 1

2010 ◽  
Vol 206 (2) ◽  
pp. 171-181 ◽  
Author(s):  
Sanhua Leng ◽  
Wenshuo Zhang ◽  
Yanbin Zheng ◽  
Ziva Liberman ◽  
Christopher J Rhodes ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Molecular Mechanism ◽  
High Glucose ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Insulin Receptor Substrate ◽  
Glycogen Synthase Kinase 3Β ◽  
Proteasome Degradation ◽  
Protein Levels

High glucose (HG) has been shown to induce insulin resistance in both type 1 and type 2 diabetes. However, the molecular mechanism behind this phenomenon is unknown. Insulin receptor substrate (IRS) proteins are the key signaling molecules that mediate insulin's intracellular actions. Genetic and biological studies have shown that reductions in IRS1 and/or IRS2 protein levels are associated with insulin resistance. In this study we have shown that proteasome degradation of IRS1, but not of IRS2, is involved in HG-induced insulin resistance in Chinese hamster ovary (CHO) cells as well as in primary hepatocytes. To further investigate the molecular mechanism by which HG induces insulin resistance, we examined various molecular candidates with respect to their involvement in the reduction in IRS1 protein levels. In contrast to the insulin-induced degradation of IRS1, HG-induced degradation of IRS1 did not require IR signaling or phosphatidylinositol 3-kinase/Akt activity. We have identified glycogen synthase kinase 3β (GSK3β or GSK3B as listed in the MGI Database) as a kinase required for HG-induced serine332 phosphorylation, ubiquitination, and degradation of IRS1. Overexpression of IRS1 with mutation of serine332 to alanine partially prevents HG-induced IRS1 degradation. Furthermore, overexpression of constitutively active GSK3β was sufficient to induce IRS1 degradation. Our data reveal the molecular mechanism of HG-induced insulin resistance, and support the notion that activation of GSK3β contributes to the induction of insulin resistance via phosphorylation of IRS1, triggering the ubiquitination and degradation of IRS1.

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