With both AMPK and PKA, phosphorylation of PDE4B in the presence of [-32P] ATP was additive, suggesting the presence of distinct phosphorylation sites. of PDE protein was reached (Fig. 5c). With both AMPK and PKA, phosphorylation of PDE4B in the presence of [-32P] ATP was additive, suggesting the presence of unique phosphorylation sites. After maximal phosphorylation by AMPK and [-32P] ATP, followed by trypsin digestion and peptide separation by HPLC, three AMPK phosphorylation sites were recognized by liquid chromatography-coupled tandem mass spectrometry (LCCMS/MS) in the major radiolabelled peaks as Ser118, Ser125 and Ser304 (Fig. 5b). Ser118 is located in the upstream conserved regulatory region 1 of PDE4B, and was also phosphorylated by PKA, in agreement with previous reports of phosphorylation at this site leading to activation of long PDE4 isoforms32,32. Ser125 is also situated in upstream conserved regulatory region 1, while Ser304 corresponds to Ser245 located in the catalytic domain name of PDE4D9 (ref. 34). The sequences surrounding Ser118, Ser125 and Ser304 are well conserved in vertebrate PDE4 orthologues (Supplementary Fig. 3A) and in the different mouse PDE4 isoforms (Supplementary Fig. 3B). When Ala residues were launched by site-directed mutagenesis to replace each Ser, the stoichiometry of phosphorylation by AMPK decreased by 40C60% for the purified mutant recombinant proteins compared with wild-type PDE4B (Fig. 5c and Supplementary Fig. 4). phosphorylation of wild-type PDE4B by AMPK increased the and purified. PDE protein was phosphorylated for 1?h with purified recombinant activated AMPK and/or purified PKA catalytic subunits and [-32P] ATP, and analysed by SDSCPAGE followed by Coomassie blue staining and APNEA phosphorimaging for quantification (a,c). In b, PDE was phosphorylated for 1?h with recombinant activated AMPK and [-32P]. Phosphorylation sites were recognized by LCCMS/MS after trypsin digestion and radioactive peak separation by high-performance liquid chromatography (HPLC). The phosphorylation sites that were recognized are underlined in the TRADD right hand panel. In d and e, recombinant APNEA PDE was phosphorylated as above but with non-radioactive ATP for PDE assay as indicated. In d, individual determinations of (Fig. 6a). Following immunoprecipitation of endogenous PDE4B from intact hepatocytes incubated with 991 or phenformin and immunoblotting, phosphorylation increased at the three main sites we recognized (Fig. 6b), although some basal phosphorylation was seen in control-incubated hepatocytes. In hepatocytes from wild-type mice incubated with increasing concentrations of 991 or phenformin up to maximal doses, phosphorylation of AMPK, ACC and Raptor was increased, and this increase was completely abrogated or reduced in hepatocytes from AMPK 1?/?2LS?/? mice (Fig. 6c). Again, although some basal PDE4B phosphorylation at the activating site Ser304 was seen in untreated hepatocytes, incubation of hepatocytes with the highest doses of 991 and phenformin led to significant increases in PDE4B Ser304 phosphorylation, which were lost in hepatocytes from AMPK 1?/?2LS?/? mice (Fig. 6c). Basal PDE4B Ser304 phosphorylation, which was also apparent in hepatocytes lacking AMPK, suggests that kinase(s) other than AMPK could phosphorylate PDE4B. It is noteworthy that users of the AMPK-related salt-inducible kinase (SIK) family were shown to be involved in the regulation of hepatic gluconeogenesis35,36, and SIK1 was recently reported to activate mouse PDE4D in pancreatic -cells via phosphorylation of Ser136 (ref. 37), the residue corresponding to Ser125 of PDE4B recognized here. Open in a separate window Physique 6 AMPK activation prospects to PDE4B phosphorylation in intact hepatocytes.In a, wild-type (WT) or mutant recombinant mouse liver PDE4B was incubated for 1?h with non-radioactive ATP in the presence (+) or absence (?) of recombinant activated AMPK. Proteins (0.1?g) were seperated by SDSCPAGE for immunoblotting with the indicated antibodies. In b and c, mouse hepatocytes from either WT (b) or both WT and AMPK 1?/?2LS?/? mice (c) were serum-starved overnight and incubated for 1?h with the indicated concentrations of 991 or phenformin. The cells were collected and lysed for immunoblotting with the indicated antibodies, except for PDE4B, which was immunoprecipitated as explained in the Methods section, before immunoblotting. In c, phosphorylation levels of AMPK and its targets ACC, Raptor and PDE4B were quantified by densitometry and expressed relative to the corresponding total protein levels or GAPDH before normalization as indicated. Representative immunoblots are shown and for blot quantification in c, the values are meanss.e.m. for (Fig. 5aCc), resulting in an increase in and 0.5?mM CaCl2) by perfusion through the substandard vena cava at a rate of 5?ml?min?1 as described18. The liver was removed and hepatocytes were extracted in attachment medium (DMEM supplemented with 1?g?l?1 glucose, 4?mM glutamine, 1?mM pyruvate, penicillin/streptomycin, 10% (v/v) FBS, 10?nM insulin, 200?nM triiodothyronine (T3) and 500?nM dexamethasone). After filtering through a 100-m mesh cell strainer (BD Falcon), cells were pelleted (50 2?min) and APNEA resuspended in attachment medium for.