Protein Phosphatase-2Cα as a Positive Regulator of Insulin Sensitivity through Direct Activation of Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes

Abstract

During differentiation, expression of protein phosphatase-2C␣ (PP2C␣) is increased in 3T3-L1 adipocytes.To elucidate the role of PP2C␣ in insulin signaling, we overexpressed wild-type (WT) PP2C␣ by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2C␣-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2C␣-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2C␣(R174G) did not produce such effects. Furthermore, overexpression of PP2C␣-WT (but not PP2C␣(R174G)) decreased the 32 P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2C␣ activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2C␣ by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2C␣ antibody or PP2C␣ small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2C␣ is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.Phosphorylation state is regulated by both protein kinase and protein phosphatase and is critical for the regulation of cellular functions such as cell growth, differentiation, and metabolism (1, 2). The protein phosphatases of eukaryotic cells are structurally and functionally diverse enzymes that can be divided into two distinct families (serine/threonine phosphatases and protein-tyrosine phosphatases) based on their specificities for phosphoamino acids (3, 4). Serine/threonine phosphatases are further classified into four major groups (protein phosphatase (PP) 1 -1, PP2A, PP2B, and PP2C␣) depending on their substrate specificities, bivalent cation dependences, and sensitivities to various inhibitor molecules such as protein inhibitor-1 and -2 and the tumor promoter okadaic acid (3, 5).The role of protein-tyrosine phosphatases in insulin signaling has been well characterized in several recent studies (6 -11). As does tyrosine phosphorylation, serine/threonine phosphorylation plays important roles in insulin signal transduction. A major role in the negative regulation of insulin action is attributed to agents that enhance serine/threonine phosphorylation of the receptor itself or its downstream effectors such as insulin receptor substrate-1 (IRS-1). Serine/threonine phosphorylation of such molecules induces insulin resistance. Furthermore, Akt and atypical protein kinase C (PKC ) are serine/threonine kinases, and their kinase activities are regulated through the serine/threo...

Human adenovirus Ad-36 induces adipogenesis via its E4 orf-1 gene

“…Increased proliferation, differentiation and activation of PI3K in adipocytes results in greater insulin sensitivity. 8,37 Therefore, the ability of Ad-36 to induce adipogenesis may be important for influencing insulin sensitivity. Indeed, Ad-36 infection of rats increased by several fold adipose tissue expression of many adipogenic genes including PPARg and C/EBP-b, reduced fasting insulin levels to 54% and significantly improved the HOMA index.…”

Delayed Re-epithelialization in Ppm1a Gene-deficient Mice Is Mediated by Enhanced Activation of Smad2

“…Overactivation of Smad2 in Ppm1a-deficient Mice-Previous studies have shown that Ppm1a could modulate Smad and PI3K/Akt signaling pathways, both of which are involved in wound healing process and keratinocyte migration (9,12,19,21,31). So we checked the expression levels of p-Akt and p-Smad2/3 at wound edge from Ppm1a-null and control mice by Western blot.…”

Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

“…This result support our assumption regarding the indirect inhibition of PI3K by PRKCA, since, if PRKCA directly phosphorylates PI3K leading to its inhibition, the phosphatases inhibition would result in even greater PI3K inhibition, and this was not the case. An opposite regulation of PI3K by phosphatases was found in 3T3-L1 adipocytes in which overexpression of PP2Ca resulted in direct activation of PI3K by dephosphorylating the inhibitory site Ser608 in PI3K regulatory subunit -p85 (Yoshizaki et al 2004). The fact that PI3K has many phosphorylation sites which are activatory or inhibitory (Yoshizaki et al 2004, together with the established existence of variety of modulators, targeting and regulatory subunits that function as complexes with phosphatases and regulate their specific activity (Cohen 2002), points to the possibility that different phosphatases have specific sites for dephosphorylation on PI3K.…”

“…An opposite regulation of PI3K by phosphatases was found in 3T3-L1 adipocytes in which overexpression of PP2Ca resulted in direct activation of PI3K by dephosphorylating the inhibitory site Ser608 in PI3K regulatory subunit -p85 (Yoshizaki et al 2004). The fact that PI3K has many phosphorylation sites which are activatory or inhibitory (Yoshizaki et al 2004, together with the established existence of variety of modulators, targeting and regulatory subunits that function as complexes with phosphatases and regulate their specific activity (Cohen 2002), points to the possibility that different phosphatases have specific sites for dephosphorylation on PI3K. Therefore, we assume that PPP1 dephosphorylates PI3K on 'positive'/stimulatory-phospho-site, leading to PI3K inhibition.…”