3-Phosphoinositide-dependent Protein Kinase 1, an Akt1 Kinase, Is Involved in Dephosphorylation of Thr-308 of Akt1 in Chinese Hamster Ovary Cells

Abstract

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using antiphosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulinstimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulinstimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.Akt, also known as protein kinase B or Rac-PK, is a Ser/Thr kinase that was originally identified as a transforming oncogene in a retrovirus from a spontaneous thymoma in an AKR mouse (1). Activation of Akt1 by growth factors, such as insulin and IGF-1, 1 has been shown to be necessary for various cellular processes including cell growth, differentiation, metabolism, and apoptosis (2-6). To date, two potential in vivo substrates of Akt1 have been identified, namely glycogen synthase kinase-3 (GSK3) and BAD (4). GSK3 is inhibited by Akt1, which is thought to contribute to the insulin-induced dephosphorylation (activation) of glycogen synthase and protein synthesis initiation factor eIF2B and thereby to the stimulation of glycogen synthesis and protein synthesis (4). In addition, one of the cellular targets that Akt1 may phosphorylate to protect cells from apoptosis is BAD (7,8). This protein, in its dephosphorylated form, interacts with the Bcl family member BclXL and induces apoptosis of some cells; however, BAD, which is phosphorylated by Akt1, dissociates from BclXL, instead of interacting with 14-3-3, to prevent apoptosis (9). Furthermore, in transfection-based experiments, constitutively active Akt1 also mimics other actions of insulin, such as the enhancement of glucose uptake in 3T3-L1 adipocytes (10) and L6 myotubes (11) that results in the translocation of GLUT4 from an intracellular compartment to the plasma membrane. Although several lines of evidence ...

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

“…However, it could also reflect differences in the activities of, or accessibilities by various phosphatases to the different activation loops. Surprisingly little is known about phosphatases which act on the activation loop residues of AGC kinases, with limited evidence implicating protein phosphatase 2A (PP2A) for PKB/Akt and PKC isoforms [40,41]. Given the large disparity seen here for dephosphorylation of different activation loop residues, further work in this area is warranted.…”

Quantitative analysis of Akt phosphorylation and activity in response to EGF and insulin treatment

“…We do not see any evidence of differential S473 versus T308 early phase phosphorylation, but this may be due to a lack in temporal resolution at early times in our experiment. Although the phosphorylation of T308 and S473 is expected to be tightly coupled, several studies have shown that the dephosphorylation of these two sites is uncoupled [10,11]. In particular, prior studies using the shellfish toxin okadaic acid suggest that phosphatase activity at the T308 site is not connected to dephosphorylation at the S473 site, with further work suggesting that the phosphatase PP2A is responsible for T308 dephosphorylation [10,11,17].…”

“…Although the phosphorylation of T308 and S473 is expected to be tightly coupled, several studies have shown that the dephosphorylation of these two sites is uncoupled [10,11]. In particular, prior studies using the shellfish toxin okadaic acid suggest that phosphatase activity at the T308 site is not connected to dephosphorylation at the S473 site, with further work suggesting that the phosphatase PP2A is responsible for T308 dephosphorylation [10,11,17]. Gao et al recently showed that a novel phosphatase, PHLPP, is responsible for dephosphorylating the S473 site [10].…”

“…The T308 and S473 phosphorylation sites have been the primary focus of a large number of biochemical studies into Akt's mechanism of activation. Salient results include the finding that phosphorylation on both the S473 and T308 sites is necessary for full kinase activation in response to insulin [with S473 phosphorylation alone inducing no activity and T308 phosphorylation alone inducing approximately one-third maximum activity [8]], that S473 phosphorylation precedes and promotes T308 phosphorylation [9], and that the dephosphorylation of the two sites often occurs differentially and through separate phosphatases [10,11]. Facile measurement of these two sites using western blot or immunostaining techniques has been enabled by the availability of phospho-specific antibodies against T308 and S473.…”

“…Threonine 72 and serine 246 have also recently been identified as autophosphorylated sites that regulate kinase activity [14]. Furthermore, uncoupling of T308 phosphorylation and kinase activity after initial kinase activation has been reported in response to insulin [11].…”