Phosphorylation of a cAMP-specific phosphodiesterase (HSPDE4B2B) by mitogen-activated protein kinase

被引：40

作者：

Lenhard, JM ^{[1
]}

Kassel, DB ^{[1
]}

Rocque, WJ ^{[1
]}

Hamacher, L ^{[1
]}

Holmes, WD ^{[1
]}

Patel, I ^{[1
]}

Hoffman, C ^{[1
]}

Luther, M ^{[1
]}

机构：

[1] GLAXO INC, RES INST, RES TRIANGLE PK, NC 27709 USA

来源：

BIOCHEMICAL JOURNAL | 1996年 / 316卷

关键词：

D O I：

10.1042/bj3160751

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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), HSPDE4BZB(81-564) and HSPDE4B2B(152-528) showed that phosphorylation occurred predominantly on Ser(487) and Ser(489). The stoicheiometry of phosphorylation 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 of purified HSPDE4B2B(152-528) expressed in Escherichia coli. Radiolabelling experiments with P-32 revealed that phosphorylation of HSPDE4B2B(152-528) expressed in Sf9 cells was abolished when Ser(487) and Ser(489) were mutated to alanines. Analysis of the amino acid sequence revealed that Ser(487) and Ser(489) 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 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 kinase-phosphorylated E. coli expressed HSPDE4B2B(152-528) exclusively at Ser(487). These results suggest that phosphodiesterases could contribute to the cross-talk between the cAMP and MAP kinase signalling pathways.

引用

页码：751 / 758

页数：8

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