Ceramide Disables 3-Phosphoinositide Binding to the Pleckstrin Homology Domain of Protein Kinase B (PKB)/Akt by a PKCζ-Dependent Mechanism

Abstract

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKC, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKC interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKC, leading to phosphorylation of the PKB-PH domain on Thr 34 . This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP 3 even in the presence of a ceramide-activated PKC and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKC and a kinase-dead PKC both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKC-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.Protein kinase B (PKB), also known as c-Akt, is a serine/ threonine kinase that has been implicated in the control of diverse cellular functions, including glucose metabolism, gene transcription, cell proliferation, and apoptosis (16,27,34,48). Three PKB isoforms (␣, ␤, and ␥) have been identified, and these can be activated rapidly in response to insulin and growth factors in a phosphoinositide 3-kinase (PI3K)-dependent manner. PI3K activation results in the increased production of 3-phosphoinositides, e.g., phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol 3,4-bisphosphate, which play a key role in the recruitment of PKB to the plasma membrane (5). The N-terminal domain of all three PKB isoforms contains a pleckstrin homology (PH) domain, which is considered critical in allowing the kinase to interact with 3-phosphoinositides and possibly other signaling proteins (13,16,19). Binding of 3-phosphoinositides to the PH domain of PKB is also thought to induce conformational changes in the kinase that expose two key regulatory sites, Thr 308 and Ser 473(3), allowing them to be phosphorylated by two upstream kinases. One of these, 3-phosphoinositide-dependent kinase-1 (PDK1), phosphorylates Thr 308 (4, 44), whereas the identity of the second kinase that phosphorylates Ser 473 (putatively termed PDK2) remains unknown, although a number of potential candidates have recently been proposed (for a review, see reference 15).The activation of PKB elicited by insulin and g...

Involvement of sphingosine 1-phosphate in palmitate-induced insulin resistance of hepatocytes via the S1P2 receptor subtype

“…For instance, ceramides directly activate a PKCζ isoform that inhibits the translocation of Akt. Additionally ceramides stimulate the activity of cytosolic protein phosphatase, which accounts for the dephosphorylation of Akt [10].…”

The Par6α/aPKC complex regulates Akt1 activity by phosphorylating Thr34 in the PH-domain

“…Interestingly, some of the serine/threonine kinases that negatively regulate IRS proteins are also insulin-sensitive, like e. g. IKKβ (I-kappa B kinase β), JNK (c-Jun N-terminal kinase) and PKCζ, thereby providing negative feedback loops in insulin action (Zick, 2004). The role of Akt1-T34 as a target of aPKC has been identified in L6 myotubes stimulated with ceramide (Powell et al, 2003), a sphingolipid enriched in skeletal muscle of insulin-resistant subjects (Adams et al, 2004). In C2C12 cells, ceramide also caused Akt1 down-regulation, but this effect was independent of an increased aPKC activity and relied on a protein phosphatase 2A-like activity dephosphorylating Akt1 (Cazzolli et al, 2001).…”

“…The T34-phosphosite in the PH-domain of Akt1 is targeted by ceramide-activated aPKC and responsible for inhibition of insulin-induced Akt1 activation (Powell et al, 2003). As ceramide is discussed as a mediator of insulin resistance in skeletal muscle (Adams et al, 2004), we wanted to know whether Par6α activated aPKC use the same mechanism to regulate Akt1.…”

“…Saturated nonesterified fatty acids (NEFAs) or ceramide enhance the formation of the PKCζ-Akt1 complex (Bourbon et al, 2002;Powell et al, 2004), and PKCζ then phosphorylates the T34 residue in the amino-terminal pleckstrin homology (PH) domain of Akt1. This in turn reduces phosphatidylinositol-3,4,5-triphosphate binding of Akt1 and down-regulates Akt1 activation (Powell et al, 2003). As both, elevated plasma NEFAs and increased skeletal muscle ceramide content reflect typical features of type 2 diabetes (Summers et al, 2005), aPKC-dependent Akt1-T34 phosphorylation may be relevant for development of insulin resistance.…”

A novel functional polymorphism (?336A/G) in the promoter of the partitioning-defective protein-6? gene is associated with increased glucose tolerance and lower concentrations of serum non-esterified fatty acids

“…Firstly, aPKCs are able to phosphorylate insulin receptor substrate-1 (IRS-1) on serine residues, thereby downregulating the recruitment and activation of phosphatidylinositol-3-kinase (PI3K) by IRS-1, especially under prolonged stimulation with insulin [3][4][5]. Secondly, when stimulated with ceramide, aPKCs directly inhibit protein kinase B(α) [6], one of the key molecules required for insulin-dependent glucose transport and glycogen synthesis [7,8].…”