Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation

The tumor suppressor p53 is commonly inhibited under conditions in which the phosphatidylinositide 3'-OH kinase/protein kinase B (PKB) Akt pathway is activated. Intracellular levels of p53 are controlled by the E3 ubiquitin ligase Mdm2. Here we show that PKB inhibits Mdm2 self-ubiquitination via phosphorylation of Mdm2 on Ser(166) and Ser(188). Stimulation of human embryonic kidney 293 cells with insulin-like growth factor-1 increased Mdm2 phosphorylation on Ser(166) and Ser(188) in a phosphatidylinositide 3'-OH kinase-dependent manner, and the treatment of both human embryonic kidney 293 and COS-1 cells with phosphatidylinositide 3'-OH kinase inhibitor LY-294002 led to proteasome-mediated Mdm2 degradation. Introduction of a constitutively active form of PKB together with Mdm2 into cells induced phosphorylation of Mdm2 at Ser(166) and Ser(188) and stabilized Mdm2 protein. Moreover, mouse embryonic fibroblasts lacking PKBalpha displayed reduced Mdm2 protein levels with a concomitant increase of p53 and p21(Cip1), resulting in strongly elevated apoptosis after UV irradiation. In addition, activation of PKB correlated with Mdm2 phosphorylation and stability in a variety of human tumor cells. These findings suggest that PKB plays a critical role in controlling of the Mdm2.p53 signaling pathway by regulating Mdm2 stability.

Advances in protein kinase B signalling: AKTion on multiple fronts

The role of the serine/threonine protein kinase B (PKB, also known as Akt) is becoming increasingly more evident to researchers investigating diverse cellular processes such as glucose uptake, cell-cycle progression, apoptosis and transcriptional regulation. New roles for PKB/Akt have been described in various organisms and biological processes. From the regulation of ovarian ecdysteroid production in the humble mosquito (Aedes aegypti), through the seasonal, tissue-specific regulation of PKB/Akt during the hibernation of yellow-bellied marmots (Marmota flaviventris), to the control of glucose metabolism and insulin signalling in the mouse (Mus musculus), our knowledge of the function of this protein kinase has expanded greatly in recent years. Significant advances in all aspects of PKB/Akt signalling have occurred in the past 2 years, including biological insights, novel substrates and newly discovered regulatory mechanisms of PKB/Akt. Collectively, these data expand the current models of PKB/Akt signalling and highlight potential directions for PKB/Akt research in the future.

Ten years of protein kinase B signalling: a hard Akt to follow

It is ten years since the publication of three papers describing the cloning of a new proto-oncogene serine/threonine kinase termed protein kinase B (PKB)/Akt. Key roles for this protein kinase in cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration are now well established. The explosion of publications involving PKB/Akt in the past three years emphasizes the high level of current interest in this signalling molecule. This review focuses on tracing the characterization of this kinase, through the elucidation of its mechanism of regulation, to its role in regulating physiological and pathophysiological processes,to our current understanding of the biology of PKB/Akt, and prospects for the future.

Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased similar to6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.

Insulin-stimulated protein kinase B phosphorylation on Ser-473 is independent of its activity and occurs through a staurosporine-insensitive kinase

Full activation of protein kinase B (PKB, also called Akt) requires phosphorylation on two regulatory sites, Thr-308 in the activation loop and Ser-473 in the hydrophobic C-terminal regulatory domain (numbering for PKB alpha /Akt-1), Although 3 ' -phosphoinositide-dependent protein kinase 1 (PDK1) has now been identified as the Thr-308 kinase, the mechanism of the Ser-473 phosphorylation remains controversial. As a step to further characterize the Ser-473 kinase, we examined the effects of a range of protein kinase inhibitors on the activation and phosphorylation of PKB. We found that staurosporine, a broad-specificity kinase inhibitor and inducer of cell apoptosis, attenuated PKB activation exclusively through the inhibition of Thr-308 phosphorylation, with Ser-473 phosphorylation unaffected. The increase in Thr-308 phosphorylation because of overexpression of PDK1 was also inhibited by staurosporine, We further show that staurosporine (CGP 39360) potently inhibited PDK1 activity in vitro with an IC50 of similar to0.22 muM. These data indicate that agonist-induced phosphorylation of Ser-473 of PKB is independent of PDK1 or PKB activity and occurs through a distinct Ser-473 kinase that is not inhibited by staurosporine, Moreover, our results suggest that inhibition of PKB signaling is involved in the proapoptotic action of staurosporine.

Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase

Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER)(1). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes(2). Controversy exists, however, concerning whether ER has a role outside the nucleus(3), particularly in mediating the cardiovascular protective effects of oestrogen(4). Here we show that the ER isoform, ER alpha, binds in a ligand-dependent manner to the p85 alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ER alpha-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ERa are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85 alpha, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ERa with PI(3)K.

Multiplexing Sensory Molecules Map Protons Near Micellar Membranes

Hi-fi mapping: Multiplexing fluorescent sensors that simultaneously target proton concentration and polarity move to micellar nanospaces, self-regulate their positions, and report their pKa values and wavelengths, which are controlled by their local environments. Such sensory functions enable maps of proton gradients near micellar membranes to be drawn.