Phosphorylation of a cAMP-specific phosphodiesterase (HSPDE4B2B) by mitogen-activated protein kinase.
A cAMP-specific phosphodiesterase, HSPDE4B2B, was found to be phosphorylated when expressed in Sf9 cells or yeast. Deletion of amino acids 81-151 and 529-564 had no effect on the phosphorylation of HSPDE4B2B. Mass spectrometric analysis of purified HSPDE4B2B(1-564). HSPDE4B2B(81-564) and HSPDE4B2B(152-528) showed that phosphorylation occurred predominantly on Ser487 and Ser489. The stoicheiometry of phosphorylation was 1.2:1 (Ser487:Ser487, 489). There was no 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 of purified HSPDE4B2B(152-528) expressed in Escherichia coli. Radiolabelling experiments with 32P revealed that phosphorylation of HSPDE4B2B(152-528) expressed in Sf9 cells was abolished when Ser487 and Ser489 were mutated to alanines. Analysis of the amino acid sequence revealed that Ser487 and Ser489 of HSPDE4B2B conform to the consensus motifs for phosphorylation by mitogen-activated protein kinase (MAP kinase) and casein kinase II respectively. Kinetic experiments in vitro showed that MAP kinase-phosphorylated E.coli expressed and purified HSPDE4B2B(151-528) with a K(m) of 63 microM and a Vmax of 3.0 mumol/min per mg. In comparison, MAP kinase phosphorylated myelin basic protein with a Km of 26.0 microM and a Vmax of 5.5 mumol/min per mg under the same conditions. Using MS and mutational analysis we found that MAP kinase-phosphorylated E. coli expressed HSPDE4B2B(152-528) exclusively at Ser487. These results suggest that phosphodiesterases could contribute to the cross-talk between the cAMP and MAP kinase signalling pathways.
Lenhard
J M
JM
Glaxo Research Institute, Research Triangle Park, NC 27709, USA.
Kassel
D B
DB
Rocque
W J
WJ
Hamacher
L
L
Holmes
W D
WD
Patel
I
I
Hoffman
C
C
Luther
M
M
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D012694:Serine; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D012694:Serine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000409:Alanine