Protein Kinase C βII Regulates Akt Phosphorylation on Ser-473 in a Cell Type- and Stimulus-specific Fashion

Abstract

Akt ‫؍(‬ protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKC␤II, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKC␤ is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKC␤II appears to work as a cell type-and stimulusspecific PDK2.Akt is a subfamily of the AGC serine/threonine kinases consisting of Akt1/PKB␣, Akt2/PKB␤, and Akt3/PKB␥. These kinases have an N-terminal pleckstrin homology domain followed by a C-terminal kinase catalytic domain. Numerous studies implicate Akt in survival, proliferation, and glucose metabolism (reviewed in Refs. 1-3). Akt activation requires the recruitment of the enzyme to the plasma membrane through the interaction of the pleckstrin homology domain with plasma membrane-bound lipid products of phosphatidylinositol 3-kinase, i.e. phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (4 -7). Membrane-bound Akt is then phosphorylated at two sites for full activation (8); Thr-308 in the activation loop of the kinase domain is phosphorylated by PDK1 1 (5, 7, 9) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been controversial (10), although several PDK2 candidates including integrin-linked kinase (ILK) (11, 12) and autophosphorylation (13) have been proposed. PKC is also a subfamily of the AGC serine/threonine kinases, which are involved in proliferation, differentiation, metabolism, and cell-type specific functions (14, 15). Based on structural features and cofactor requirements, PKC isoforms are classified into three categories; conventional or classical isoforms (cPKCs: ␣, ␤I, ␤II, and ␥) depend on diacylglycerol (DAG) and Ca 2ϩ for their activation, novel isoforms (nPKCs: ␦, ⑀, , and ) depend on DAG, but not Ca 2ϩ , and atypical isoforms (atypical PKC isoforms: and /) do not require either DAG or Ca 2ϩ .Cross-linking of high-affinity IgE receptors (Fc⑀RI) expressed on the surface of mast cells activates Akt (16) and various PKC isoforms including PKC␤I and PKC␤II (17). Importantly, Akt in mast cells is involved in Fc⑀RI-induced production of cytokines such as IL-2 through the activation of such trans...

14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells

“…Akt phosphorylation at Ser473 has been extensively studied as a correlate for Akt activity; [11][12][13]24,25 however, the mechanisms controlling the phosphorylation of Thr308 are rarely assessed. Thus, the machinery involved in the phosphorylation of Akt at the residue Thr308 during colitis was investigated.…”

“…7 PDK1 activation requires auto-phosphorylation of the residue Ser241 and is regulated by its association with several proteins including: HSP90, 8 RSK2 9 and 14-3-3 family members. 10 By contrast, the phosphorylation of Akt at Ser473 is mediated mainly by mTORC2, 11 but can also be achieved by other kinases, including integrin-linked kinase (ILK), 12 protein kinase C (PKCε) 13 and by auto-phosphorylation. 14 Phosphorylation of Thr308 has been proposed to be a critical regulatory event for Akt activation.…”

Specific protein kinase C isoforms as transducers and modulators of insulin signaling

“…The phosphorylation of TLS-FUS, a nuclear transcription factor that also functions in splicing, demonstrates the versatility of PKCβII functions [94,95]. The observation that PKCβII acts to phosphorylate Akt on Ser473 and acts as a PDK2 (phosphoinositide dependent kinase type 2) activity [96], underscores the role of PKCβII in signaling pathways.…”

Akt–PDK1 Complex Mediates Epidermal Growth Factor-induced Membrane Protrusion through Ral Activation

“…PDK1 phosphorylates Akt at Thr 308 , which causes a charge-induced conformational change, allowing Akt for substrate binding and an increased rate of catalysis. Akt is further activated by the phosphorylation of Ser 473 , which can be catalyzed by various kinases, including PDK2, DNA-PK, mammalian target of rapamysin, rictor complex, integrin linked kinase, and protein kinase C␤II (Feng et al, 2004;Kawakami et al, 2004;Fayard et al, 2005;Sarbassov et al, 2005 It has been demonstrated that PI3K plays an essential role in the epidermal growth factor (EGF)-induced membrane protrusion by regulating not only Akt (Higuchi et al, 2001;Nishita et al, 2004;Hafizi et al, 2005) but also Rac and Ral GTPases (Tian et al, 2002;Gavard et al, 2004;Nishita et al, 2004;Takaya et al, 2004). However, the mechanism by which Akt regulates the cytoskeletal remodeling and the relationship to Ral and Rac has been still remains elusive.…”