PHOSPHORYLATION OF A CAMP-SPECIFIC PHOSPHODIESTERASE (HSPDE4B2B) BY MITOGEN-ACTIVATED PROTEIN-KINASE

A cAMP-specific phosphodiesterase, HSPDE4B2B, was found to be phosphor ylated when expressed in Sf9 cells or yeast. Deletion of amino acids 8 1-151 and 529-564 had no effect on the phosphorylation of HSPDE4B2B. M ass spectrometric analysis of purified HSPDE4B2B(1-564), HSPDE4BZB(81- 564) and HSPDE4B2B(152-528) showed that phosphorylation occurred predo minantly on Ser(487) and Ser(489). The stoicheiometry of phosphorylati on was 1.2:1 (Ser(487),Ser(487,489)). There was Ilo evidence by MS for a non-phosphorylated form of HSPDE4B2B(81-564) or HSPDE4B2B(152-528) when expressed in Sf9 cells. There was no detectable phosphorylation o f purified HSPDE4B2B(152-528) expressed in Escherichia coli. Radiolabe lling experiments with P-32 revealed that phosphorylation of HSPDE4B2B (152-528) expressed in Sf9 cells was abolished when Ser(487) and Ser(4 89) were mutated to alanines. Analysis of the amino acid sequence reve aled that Ser(487) and Ser(489) of HSPDE4B2B conform to the consensus motifs for phosphorylation by mitogen-activated protein kinase (MAP ki nase) and casein kinase II respectively. Kinetic experiments in vitro showed that MAP kinase-phosphorylated E. coli expressed and purified H SPDE4B2B(151-528) with a K-m of 63 mu M and a V-max of 3.0/mu mol/min per mg. In comparison, MAP kinase phosphorylated myelin basic protein with a K-m of 26.0 mu M and a V-max of 5.5 mu mol/min per mg under the same conditions. Using MS and mutational analysis we found that MAP k inase-phosphorylated E. coli expressed HSPDE4B2B(152-528) exclusively at Ser(487). These results suggest that phosphodiesterases could contr ibute to the cross-talk between the cAMP and MAP kinase signalling pat hways.