With the use of immunoprecipitation of endogenous or epitope-tagged recombinant proteins, we have shown that insulin induces phosphorylation of PDE3B in 3T3-L1 adipocytes. Insulin-induced phosphorylation of PDE3B was prevented by expression of a dominant-negative mutant of the regulatory subunit of PI 3-kinase (Δp85), and expression of a constitutively active mutant of PI 3-kinase (Myr-p110) stimulated phosphorylation of PDE3B in the absence of insulin. Furthermore, a mutant Akt in which the sites of ligand-induced phosphorylation were replaced by alanine (Akt-AA), which prevents insulin activation of Akt (23
), abolished insulin-induced phosphorylation of PDE3B, and expression of a constitutively active mutant of Akt (Myr-Akt) resulted in phosphorylation of PDE3B in quiescent 3T3-L1 adipocytes. These data suggest that the PI 3-kinase–Akt pathway is necessary and sufficient for insulin-induced phosphorylation of PDE3B.
Akt-K179D or similar kinase-deficient mutants of Akt that contain a substitution at Lys179
in the kinase domain have little effect on growth factor-induced activation of Akt (23
). However, such mutants block certain biological effects that are likely mediated by Akt, including growth factor-induced phosphorylation of PHAS1 (4E-BP1) (20
), growth factor-induced phosphorylation of BAD and consequent protection of cells from apoptosis (12
), and insulin-induced activation of glycogen synthase (44
). We have now shown that Akt-K179D inhibited insulin-induced phosphorylation of PDE3B. One possible explanation for these observations is that Akt-K179D and similar kinase-deficient mutants of Akt compete with endogenous Akt for cellular substrates of the enzyme and thereby exert dominant-negative effects on various biological activities.
We have shown that Akt associates with PDE3B when these proteins are overexpressed in CHO cells. It is noteworthy that not only wild-type but kinase-deficient mutants of Akt form a complex with PDE3B, indicating that the interaction is independent of the kinase activity of Akt. This result is consistent with the finding that both wild-type and a kinase-inactive mutant of Akt make a complex with BAD (12
). Moreover, treatment of the cells with insulin or wortmannin did not affect the interaction. We do not know why Akt and PDE3B form a constitutive complex in cells whereas PDE3B primarily resides in the membrane fraction and Akt translocates from cytosol to plasma membrane in response to insulin (4
). One possibility is that overexpression of these proteins may result in “overflow” from their authentic intracellular compartments and that this overflow may cause constitutive complex formation. It remains to be investigated under what conditions endogenous PDE3B and Akt interact in intact cells.
Several proteins have been shown to be phosphorylated by Akt in vitro or in intact cells. These proteins include GSK3β, BAD, and phosphofructose-2-kinase (Fig. ) (11
). On the basis of studies with peptides derived from these proteins, it has been suggested that Akt preferentially phosphorylates substrates that conform to the sequence RXRXXS. Substitution of alanine for Ser273
of mouse PDE3B, a serine residue that resides in such a consensus motif, almost completely prevented the effect of insulin on the phosphorylation of this protein in intact cells, supporting the hypothesis that PDE3B is a physiological substrate of Akt. We also showed that PDE3B underwent phosphorylation on incubation with Akt in vitro and that, again, replacement of Ser273
, but not of other serine residues, with alanine markedly reduced the extent of Akt-mediated phosphorylation of PDE3B. These results suggest that Akt directly phosphorylates PDE3B on Ser273
FIG. 8 Alignment of amino acid sequences conforming to a motif preferentially phosphorylated by Akt in GSK3β, phosphofructose-2-kinase (PFK2), BAD, PDE3A, and PDE3B. Conserved arginine residues at positions −3 and −5 relative to the phosphorylated (more ...)
Rahn et al. (36
) showed that insulin increased incorporation of 32
P into a specific phosphopeptide generated by tryptic digestion of PDE3B that had been immunoprecipitated from 32
P-labeled rat adipocytes. They also showed that the mobility of this peptide on two-dimensional gel electrophoresis was similar to that of a tryptic peptide generated from recombinant PDE3B that had been phosphorylated by PKA in vitro. Because the latter peptide contained only one serine residue (Ser302
, which corresponds to Ser296
of the mouse enzyme), these investigators concluded that the site of insulin-induced phosphorylation in PDE3B is identical to that targeted by PKA in vitro. However, we have now shown that substitution of Ser296
of mouse PDE3B does not affect insulin-induced phosphorylation of the enzyme. In contrast, phosphorylation of PDE3B induced by isoproterenol, a reagent that increases cellular cAMP concentration and subsequently activates PKA, was markedly attenuated by substitution of Ser296
. These results suggest that Ser296
of mouse PDE3B is phosphorylated in response to isoproterenol but not insulin.
Previous studies have suggested that phosphorylation of PDE3B correlates with its catalytic activity (13
). Indeed, we have now shown that insulin activation of PDE3B was inhibited by wortmannin or by Akt-AA, both of which also prevented insulin-induced phosphorylation of PDE3B. Furthermore, the importance of phosphorylation of PDE3B for its enzymatic activity was directly evaluated with the use of various mutants of PDE3B containing substitutions for various serine residues. When expressed in 3T3-L1 adipocytes, PDE3B-S273A was not activated in response to insulin, whereas mutant PDE3B proteins containing substitutions at Ser274
were activated normally. These results indicate that phosphorylation of PDE3B on Ser273
is required for insulin-induced activation of the enzyme. The activity of PDE3B-S273A in quiescent cells was similar to that of the wild-type enzyme (data not shown), indicating that phosphorylation of Ser273
of PDE3B is not important for basal catalytic activity.
Because PKCλ, an atypical isoform of PKC, acts as a downstream effector of PI 3-kinase (1
), we examined the possible role of this enzyme in the phosphorylation and activation of PDE3B. We have recently shown that expression of λΔPD promotes glucose transport in quiescent 3T3-L1 adipocytes and that λΔNKD inhibits insulin stimulation of glucose transport (29
). Although AxCAλΔPD induced phosphorylation of PDE3B, infection with this virus did not increase the activity of PDE3B in 3T3-L1 adipocytes. Moreover, infection of the cells with AxCAλΔNKD at an MOI of 150 PFU/cell, a virus dose sufficient to inhibit almost completely insulin-induced activation of PKCλ in 3T3-L1 adipocytes (29
), affected neither phosphorylation nor activation of PDE3B induced by insulin. It is thus likely that λΔPD mediates the phosphorylation of PDE3B at residues that are not important in the regulation of its activity and that PKCλ does not contribute to the physiological signaling cascade that leads to activation of PDE3B.
Because PDE3B catalyzes the hydrolysis of cAMP, activation of this enzyme likely leads to lowering of the cellular level of cAMP. Indeed, it has been reported that insulin decreases the level of cAMP that had been elevated by cathecolamine (8
). We have now shown that insulin decreased the level of cAMP in the cells that had been treated with forskolin and that Akt-AA markedly inhibited the insulin-induced decrease in cAMP, suggesting that Akt is involved in the effect of insulin on cellular cAMP concentration.
In summary, we have identified PDE3B as a physiological substrate of Akt and demonstrated that Akt-mediated phosphorylation of Ser273
is important for insulin activation of PDE3B. Of all known PDEs, only PDE3B and PDE3A possess a serine residue located within an RXRXXS motif (Fig. ). It is thus also possible that Akt contributes to the regulation of PDE3A, which is preferentially expressed in heart and vascular smooth muscle cells and implicated in cardiac contractility and vasodilatation (6