Dephosphorylation by Default, a Potential Mechanism for Regulation of Insulin Receptor Substrate-1/2, Akt, and ERK1/2

Abstract

Protein phosphorylation is an important mechanism that controls many cellular activities. Phosphorylation of a given protein is precisely controlled by two opposing biochemical reactions catalyzed by protein kinases and protein phosphatases. How these two opposing processes are coordinated to achieve regulation of protein phosphorylation is unresolved. We have developed a novel experimental approach to directly study protein dephosphorylation in cells. We determined the kinetics of dephosphorylation of insulin receptor substrate-1/2, Akt, and ERK1/2, phosphoproteins involved in insulin receptor signaling. We found that insulin-induced ERK1/2 and Akt kinase activities were completely abolished 10 min after inhibition of the corresponding upstream kinases with PD98059 and LY294002, respectively. In parallel experiments, insulin-induced phosphorylation of Akt, ERK1/2, and insulin receptor substrate-1/2 was decreased and followed similar kinetics. Our findings suggest that these proteins are dephosphorylated by a default mechanism, presumably via constitutively active phosphatases. However, dephosphorylation of these proteins is overcome by activation of protein kinases following stimulation of the insulin receptor. We propose that, during acute insulin stimulation, the kinetics of protein phosphorylation is determined by the interplay between upstream kinase activity and dephosphorylation by default.The reversible phosphorylation of proteins has emerged as a major mechanism for the regulation of cellular signal transduction (1, 2). Disruption of protein phosphorylation/dephosphorylation has been linked to many pathological conditions, including cancer, neurodegeneration, diabetes, cystic fibrosis, asthma, and cardiovascular disease (3-5). Despite the critical importance of protein phosphorylation in signaling and disease, the underlying mechanisms that determine the kinetics of dephosphorylation remain unresolved. Two general models have been proposed to describe these mechanisms. One model posits that the phosphorylation level is determined by constitutively active phosphatases and transiently activated kinases (2, 6). This model was established based on results gathered using potent phosphatase inhibitors such as okadaic acid on intracellular proteins as well as in in vitro cell-free assays. Therefore, although it is an attractive explanation, it may not reflect mechanisms operative in intact living cells. A second model based on studies employing advanced molecular techniques conducted in both animals and genetically engineered cell lines suggests that both kinases and phosphatases are coordinately regulated (3, 7-10). However, most of these studies assessed protein dephosphorylation in the presence of upstream kinase activity; therefore, potential influences from constitutively active phosphatases may have been hidden and thus overlooked.Methods to directly measure protein dephosphorylation in vivo in the absence of upstream kinase activity are lacking. As a result, despite the aforementioned studies, we still ...

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