Insulin-Induced Phosphorylation and Activation of Cyclic Nucleotide Phosphodiesterase 3B by the Serine-Threonine Kinase Akt

Abstract

Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.Akt is a protein serine-threonine kinase that contains a pleckstrin homology domain and whose kinase domain has structural similarity with those of protein kinase C (PKC) isozymes and cyclic AMP (cAMP)-dependent protein kinase (PKA) (9, 21). Thus, Akt has also been termed protein kinase B. Akt was originally shown to be activated by growth factors such as platelet-derived growth factor and insulin, but later the enzyme was also found to be activated by cytokines and ligands for G protein-coupled receptors (21,33,34). Moreover, expression of polyomavirus middle T antigen as well as cellular stresses such as hyperosmolarity, heat shock, and fluid shear stress also induces activation of Akt (17,27,42). However, the mechanisms by which Akt is activated by these diverse stimuli are not fully understood. The activation of Akt by growth factors or cytokines is blocked by pharmacological or molecular biological inhibitors of phosphoinositide (PI) 3-kinase (7,19,24), indicating that PI 3-kinase is an upstream regulator of Akt, although PI 3-kinase-independent stimuli that induce activation of Akt also appear to exist (27,33,38).Akt is a general mediator of cell survival and protection from apoptosis (9, 21). It has also been suggested to participate in meiosis in oocytes (3), in endocytosis elicited by RAS (5), in differentiation of adipocytes (25), and in various metabolic actions of insulin (23,25,44,45). In spite of the potential importance of Akt in such diverse biological activities, only a few proteins have been identified as physiological substrates of this enzyme. The first iden...

Insulin Contributes to Fine-Tuning of the Pancreatic Beta-Cell Response to Glucagon-Like Peptide-1

“…Increased activity of PDE3B may cause rapid breakdown of cAMPs that are produced by GLP-1 activation (Härndahl et al, 2002). PDE3B is highly expressed in β-cells and can be activated by interaction with Akt (Ahmad et al, 2007;Kitamura et al, 1999). Indeed, our preliminary experiment showed that pretreatment of INS-1 cells with cilostamide, a specific inhibitor of PDE3B, significantly increased exe-4-induced cAMP accumulation and Erk phosphorylation (data not shown).…”

Novel mechanisms of the regulation of Protein kinase B in adipocytes; implications for Protein kinase A, Epac, Phosphodiesterases 3 and 4

“…In summary, these data suggest that PDE3B mediates the regulation of Ser 473 PKB phosphorylation. However, the opposite situation also appears to occur as PKB has previously been shown to regulate PDE3B phosphorylation and activation [16,35].…”

“…In adipocytes, PKB plays a crucial role in insulin-induced GLUT-4 translocation to the plasma membrane [14] and protein synthesis [15]. It also appears to be involved in the antilipolytic action of insulin through phosphorylation and activation of PDE3B [16,17].…”

Cyclic Nucleotide Phosphodiesterase 3B Is a Downstream Target of Protein Kinase B and May Be Involved in Regulation of Effects of Protein Kinase B on Thymidine Incorporation in FDCP2 Cells

“…Previous experiments have demonstrated that native RPDE3B was phosphorylated on serine 302 in intact rat adipocytes (analogous to serine 296 in MPDE3B) incubated with insulin (16), and that RPDE3B was also phosphorylated in vitro by partially purified rat adipocyte PKB (17) or rPKB (18). Available evidence suggests that phosphorylation is not required to maintain, but does increase, basal PDE activity.…”

“…More recently, in oocytes (8), rat adipocytes (16,17), and 3T3-L1 adipocytes (18), PDE3 isoforms have been suggested to be downstream targets and effectors of PKB actions. In adipocytes, insulin-induced activation of PDE3B, via P13-K-and PKB-dependent signals, is a critical component in the antilipolytic action of insulin (19 -21).…”