Role of Translocation in the Activation and Function of Protein Kinase B

Abstract

We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKB␣ was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKB␣ was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32 P labeling and mutagenesis demonstrated that m/p-PKB␣ activity was due to phosphorylation on ). Three mammalian isoforms of PKB have been identified so far, termed PKB␣, -␤, and -␥ (7-9). 2 All three isoforms contain a pleckstrin homology (PH) domain at the N terminus (10), followed by a catalytic domain related to protein kinases A and C, and a C-terminal regulatory region. PKB␣ was found to mediate insulin-and insulin-like growth factor (IGF-1)-induced cellular responses, such as the inhibition of glycogen synthase kinase-3 (11), the stimulation of glucose uptake (12), and the promotion of cell survival by inhibiting apoptosis (Ref. 13; reviewed in Refs. 14 and 15). PKB␣ is the cellular homologue of the oncogene product v-Akt encoded by the AKT8 retrovirus, which induces thymic lymphomas in mice (16). Cloning of v-akt revealed that it was created by fusion of viral Gag sequences to the N terminus of mouse PKB␣, which adds an N-terminal myristoylation signal to the oncoprotein and could account for its transforming ability (2, 17). Overexpression of PKB␣ or -␤ is associated with some human ovarian, pancreatic, and breast carcinomas (8, 18 -20).PKB␣ is activated by a variety of growth factors and phosphatase inhibitors (5, 6, 21) through a phosphorylation mechanism (21-23). The activation of PKB␣ by insulin or IGF-1 is mediated by phosphorylation of Thr 308 in the catalytic domain and Ser 473 at the C terminus (22). The phosphorylation of both sites is blocked by pretreatment of the cells with the PI3-K inhibitor wortmannin. Substitution of both regulatory sites by aspartic acid residues to mimic phosphorylation by the introduction of a negative charge, produces a constitutively active enzyme (22). This work predicted the existence of an upstream kinase(s) that phosphorylate(s) these sites, and recently a protein kinase activity was identified and purified capable of phosphorylating Thr 308 in the presence of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) or phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) (Refs. 24 and 25; reviewed in Ref. 26). The enzyme has therefore been termed 3-phosphoinositide-dependent protein kinase-1 (PDK1).The PH domain of PKB has been reported to play a role in the activation process (6), but PKB activation can also occur in its absence, depending on the agonist and the type of deletion mutants used (21,23,27). The PH domain of PKB binds PtdIns(3,4,5)P 3 and PtdIns(3,4)P...

Cloning and characterization of a nuclear S6 kinase, S6 kinase-related kinase (SRK); A novel nuclear target of Akt

“…This view is partially supported by the fact that we observe no redistribution of SRK following EGF stimulation of quiescent cells. Also, recent reports revealed that Akt, a possible upstream regulator for both SRK (Figure 4) and p70S6K (Burgering and Co er, 1995;Kohn et al, 1996), translocates from the plasma membrane to the nucleus after stimulation with mitogens, and phosphorylates nuclear as well as cytoplasmic targets (Andjelkovic et al, 1997). However, we cannot exclude the possibility that there exist separate signaling pathways which regulate SRK and p70S6K.…”

Akt2 interacts with Snail1 in the E-cadherin promoter

“…Although Akt is mainly cytosolic, a relevant fraction is also found in the nucleus, particularly of the activated form (Ahmed et al, 1993;Andjelkovic´et al, 1997). Both cytosolic and nuclear Akt activities were upregulated in Snail1-transfected cell lines (Figure 1b), an effect that, again, was more remarkable in serum-depleted cells.…”

“…Although Akt proteins localize predominantly in the cytoplasm, they also reside in the nucleus or translocate to this compartment upon stimulation (Ahmed et al, 1993;Andjelkovic´et al, 1997). In the nucleus Akt phosphorylates or interacts with proteins implicated in apoptosis resistance, a pathway where Akt has a key role (Andjelkovic´et al, 1997;Masuyama et al, 2001;Ahn et al, 2006;Lee et al, 2008).…”

“…In the nucleus Akt phosphorylates or interacts with proteins implicated in apoptosis resistance, a pathway where Akt has a key role (Andjelkovic´et al, 1997;Masuyama et al, 2001;Ahn et al, 2006;Lee et al, 2008). Moreover, Akt proteins have been involved in the process of EMT (Thiery et al, 2009).…”

CYP 2J2 and its metabolites (epoxyeicosatrienoic acids) attenuate cardiac hypertrophy by activating AMPK α2 and enhancing nuclear translocation of Akt1

“…Akt is initially activated at the cell membrane in response to stimulation by growth factors, such as insulin‐like growth factor 1 (IGF1) (Alessi et al ., 1996). After activation, Akt phosphorylates multiple cytosolic substrates and translocates to nucleus, where it is thought to regulate gene transcription (Andjelkovic et al ., 1997; Brazil et al ., 2002). Mammals express three isoforms of Akt; Akt1 and Akt2 are expressed ubiquitously, whereas Akt3 is found predominantly in the brain, kidney, and heart (Datta et al ., 1999; Masure et al ., 1999).…”