Lenhard J M, Kassel D B, Rocque W J, Hamacher L, Holmes W D, Patel I, Hoffman C, Luther M
Glaxo Research Institute, Research Triangle Park, NC 27709, USA.
Biochem J. 1996 Jun 15;316 ( Pt 3)(Pt 3):751-8. doi: 10.1042/bj3160751.
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.
一种特异性作用于环磷酸腺苷（cAMP）的磷酸二酯酶HSPDE4B2B，在Sf9细胞或酵母中表达时被发现发生了磷酸化。删除氨基酸81 - 151和529 - 564对HSPDE4B2B的磷酸化没有影响。对纯化的HSPDE4B2B(1 - 564)、HSPDE4B2B(81 - 564)和HSPDE4B2B(152 - 528)进行质谱分析表明，磷酸化主要发生在Ser487和Ser489上。磷酸化的化学计量比为1.2:1（Ser487:Ser487、489）。质谱分析没有证据表明在Sf9细胞中表达的HSPDE4B2B(81 - 564)或HSPDE4B2B(152 - 528)存在非磷酸化形式。在大肠杆菌中表达的纯化HSPDE4B2B(152 - 528)未检测到磷酸化。用32P进行的放射性标记实验表明，当Ser487和Ser489突变为丙氨酸时，在Sf9细胞中表达的HSPDE4B2B(152 - 528)的磷酸化被消除。对氨基酸序列的分析表明，HSPDE4B2B的Ser487和Ser489分别符合丝裂原活化蛋白激酶（MAP激酶）和酪蛋白激酶II磷酸化的共有基序。体外动力学实验表明，MAP激酶对在大肠杆菌中表达并纯化的HSPDE4B2B(151 - 528)进行磷酸化，其米氏常数（K(m)）为63微摩尔，最大反应速度（Vmax）为每毫克3.0微摩尔/分钟。相比之下，在相同条件下，MAP激酶对髓鞘碱性蛋白进行磷酸化时，米氏常数为26.0微摩尔，最大反应速度为每毫克5.5微摩尔/分钟。使用质谱和突变分析，我们发现MAP激酶对在大肠杆菌中表达的HSPDE4B2B(152 - 528)仅在Ser487处进行磷酸化。这些结果表明磷酸二酯酶可能参与了cAMP和MAP激酶信号通路之间的相互作用。
