|Growth hormone regulation of p85alpha expression and phosphoinositide 3-kinase activity in adipose tissue: mechanism for growth hormone-mediated insulin resistance. |
del Rincon, JP; Iida, K; Gaylinn, BD; McCurdy, CE; Leitner, JW; Barbour, LA; Kopchick, JJ; Friedman, JE; Draznin, B; Thorner, MO
Phosphoinositide (PI) 3-kinase is involved in insulin-mediated effects on glucose uptake, lipid deposition, and adiponectin secretion from adipocytes. Genetic disruption of the p85alpha regulatory subunit of PI 3-kinase increases insulin sensitivity, whereas elevated p85alpha levels are associated with insulin resistance through PI 3-kinase-dependent and -independent mechanisms. Adipose tissue plays a critical role in the antagonistic effects of growth hormone (GH) on insulin actions on carbohydrate and lipid metabolism through changes in gene transcription. The objective of this study was to assess the role of the p85alpha subunit of PI 3-kinase and PI 3-kinase signaling in GH-mediated insulin resistance in adipose tissue. To do this, p85alpha mRNA and protein expression and insulin receptor substrate (IRS)-1-associated PI 3-kinase activity were measured in white adipose tissue (WAT) of mice with GH excess, deficiency, and sufficiency. Additional studies using 3T3-F442A cells were conducted to confirm direct effects of GH on free p85alpha protein abundance. We found that p85alpha expression 1) is decreased in WAT from mice with isolated GH deficiency, 2) is increased in WAT from mice with chronic GH excess, 3) is acutely upregulated in WAT from GH-deficient and -sufficient mice after GH administration, and 4) is directly upregulated by GH in 3T3-F442A adipocytes. The insulin-induced increase in PI 3-kinase activity was robust in mice with GH deficiency, but not in mice with GH excess. In conclusion, GH regulates p85alpha expression and PI 3-kinase activity in WAT and provides a potential explanation for 1) the insulin hypersensitivity and associated obesity and hyperadiponectinemia of GH-deficient mice and 2) the insulin resistance and associated reduced fat mass and hypoadiponectinemia of mice with GH excess.
|Phosphatidylinositol 3-kinase. |
Kapeller, R and Cantley, L C
Bioessays, 16: 565-76 (1994)
Currently, a central question in biology is how signals from the cell surface modulate intracellular processes. In recent years phosphoinositides have been shown to play a key role in signal transduction. Two phosphoinositide pathways have been characterized, to date. In the canonical phosphoinositide turnover pathway, activation of phosphatidylinositol-specific phospholipase C results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the generation of two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. The 3-phosphoinositide pathway involves protein-tyrosine kinase-mediated recruitment and activation of phosphatidylinositol 3-kinase, resulting in the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. The 3-phosphoinositides are not substrates of any known phospholipase C, are not components of the canonical phosphoinositide turnover pathway, and may themselves act as intracellular mediators. The 3-phosphoinositide pathway has been implicated in growth factor-dependent mitogenesis, membrane ruffling and glucose uptake. Furthermore the homology of the yeast vps34 with the mammalian phosphatidylinositol 3-kinase has suggested a role for this pathway in vesicular trafficking. In this review the different mechanisms employed by protein-tyrosine kinases to activate phosphatidylinositol 3-kinase, and its involvement in the signaling cascade initiated by tyrosine phosphorylation, are examined.
|PI 3-kinase: structural and functional analysis of intersubunit interactions. |
Dhand, R, et al.
EMBO J., 13: 511-21 (1994)
Phosphatidylinositol (PI) 3-kinase has an 85 kDa subunit (p85 alpha) which mediates its association with activated protein tyrosine kinase receptors through SH2 domains, and an 110 kDa subunit (p110) which has intrinsic catalytic activity. Here p85 alpha and a related protein p85 beta are shown to form stable complexes with recombinant p110 in vivo and in vitro. Using a panel of glutathione S-transferase (GST) fusion proteins of the inter-SH2 region of p85, 104 amino acids were found to bind directly the p110 protein, while deletion mutants within this region further defined the binding site to a sequence of 35 amino acids. Transient expression of the mutant p85 alpha protein in mouse L cells showed it was unable to bind PI 3-kinase activity in vivo. Mapping of the complementary site of interaction on the p110 protein defined 88 amino acids in the N-terminal region of p110 which mediate the binding of this subunit to either the p85 alpha or the p85 beta proteins. The inter-SH2 region of p85 is predicted to be an independently folded module of a coiled-coil of two long anti-parallel alpha-helices. The predicted structure of p85 suggests a basis for the intersubunit interaction and the relevance of this interaction with respect to the regulation of the PI 3-kinase complex is discussed.
|Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. |
Hiles, I D, et al.
Cell, 70: 419-29 (1992)
Purified bovine brain phosphatidylinositol 3-kinase (Pl3-kinase) is composed of 85 kd and 110 kd subunits. The 85 kd subunit (p85 alpha) lacks Pl3-kinase activity and acts as an adaptor, coupling the 110 kd subunit (p110) to activated protein tyrosine kinases. Here the characterization of the p110 subunit is presented. cDNA cloning reveals p110 to be a 1068 aa protein related to Vps34p, a S. cerevisiae protein involved in the sorting of proteins to the vacuole. p110 expressed in insect cells possesses Pl3-kinase activity and associates with p85 alpha into an active p85 alpha-p110 complex that binds the activated colony-stimulating factor 1 receptor. p110 expressed in COS-1 cells is catalytically active only when complexed with p85 alpha.
|Purification and characterization of phosphoinositide 3-kinase from rat liver. |
Carpenter, C L, et al.
J. Biol. Chem., 265: 19704-11 (1990)
Phosphoinositide 3-kinase was purified 27,000-fold from rat liver. The enzyme was purified by acid precipitation of the cytosol followed by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, Mono-Q, and Mono-S columns. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified phosphoinositide 3-kinase preparation contained an 85-kDa protein and a protein doublet of approximately 110 kDa. The 85- and 110-kDa proteins focus together on native isoelectric focusing gels and are cross-linked by dithiobis(succinylamide propionate), showing that the 110- and 85-kDa proteins are a complex. The apparent size of the native enzyme, as determined by gel filtration, is 190 kDa. The 85-kDa subunit is the same protein previously shown to associate with polyoma virus middle T antigen and the platelet-derived growth factor receptor (Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 50, 1021-1029). The two proteins co-migrate on two-dimensional gels; and, using a Western blotting procedure, 32P-labeled middle T antigen specifically blots the 85-kDa protein. The purified enzyme phosphorylates phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. The apparent Km values for ATP were found to be 60 microM with phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate as the substrate. The apparent Km for phosphatidyinositol is 60 microM, for phosphatidylinositol 4-phosphate is 9 microM, and for phosphatidylinositol 4,5-bisphosphate is 4 microM. The maximum specific activity using phosphatidylinositol as the substrate is 0.8 mumol/mg/min. The enzyme requires Mg2+ with an optimum of 5 mM. Substitution of Mn2+ for Mg2+ results in only approximately 10% of the Mg2(+)-dependent activity. Physiological calcium concentrations have no effect on the enzyme activity. Phosphoinositide 3-kinase has a broad pH optimum around 7.