Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase - Involvement of serine 54 in the enzyme activation
Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase - Involvement of serine 54 in the enzyme activation
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DOI：
10.1074/jbc.271.28.16526
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发表时间：
1996-07-12
影响因子：
4.8
通讯作者：
Conti, M
中科院分区：
文献类型：
--
作者：
Sette, C;Conti, M
在大鼠甲状腺细胞中，cAMP特异性的磷酸二酯酶(PDE4D3)是由TSH通过cAMP依赖的磷酸化激活的(Sette，C.，Iona，S.，and Conti，M.(1994)J.Biol)。化学，269,9245-9252)。这种短期的激活可能参与荷尔蒙刺激的终止和/或脱敏的诱导。在此，我们进一步鉴定了PDE4D3突变体依赖蛋白激酶A(PKA)的磷酸化，并确定了参与PDE激活的磷酸化位点，部分纯化的重组大鼠PDE4D3中依赖PKA的磷酸盐掺入遵循与激活相似的时间过程，0.6mU的三磷酸腺苷和30 nM的PKA催化亚基获得了该酶的半最大激活，磷酸化改变了PDE的V-max，而不影响cAMP的K-m。磷酸化还改变了镁的需求和罗利普兰的抑制模式，溴化氰切割P-32标记的大鼠PDE4D3产生了两条或三条主要的磷酸肽带，首次表明该酶可能在无细胞体系中的多个位置被磷酸化。在PKA磷酸化的首选基序的背景下，对该PDE氨基末端的丝氨酸残基进行了定点突变。在同时含有Ser(13)->；Ala和Ser(54)-->；Ala的突变体中，依赖于PKA的P-32的掺入被最大程度地减少，证实了大鼠PDE4D3中存在不止一个磷酸化位点，而用丙氨酸替代丝氨酸13并不影响PKA的激活，Ser(54)的替代完全抑制了PKA的激活，在MA-10细胞中表达的野生型和突变的PDE4D3蛋白也得到了类似的结论，其中内源性PKA被二丁酰cAMP激活。同样，带有Ser(54)-->；Ala替代的PDE不能被完整细胞中的内源性PKA激活，这些发现支持这样的假设，即PDE4D3变体包含一个调节结构域，用于在蛋白质的氨基末端进行磷酸化，并且该结构域中的Ser(54)在激活过程中起着关键作用。
A cAMP-specific phosphodiesterase (PDE4D3) is activated in rat thyroid cells by TSH through a cAMP-dependent phosphorylation (Sette, C., Iona, S., and Conti, M. (1994) J. Biol. Chem, 269, 9245-9252). This short term activation may be involved in the termination of the hormonal stimulation and/or in the induction of desensitization. Here, we have further characterized the protein kinase A (PKA)-dependent phosphorylation of this PDE4D3 variant and identified the phosphorylation site involved in the PDE activation, The PKA-dependent incorporation of phosphate in the partially purified, re-combinant rat PDE4D3 followed a time course similar to that of activation, Half-maximal activation of the enzyme was obtained with 0.6 mu M ATP and 30 nM of the catalytic subunit of PKA, Phosphorylation altered the V-max of the PDE without affecting the K-m for cAMP. Phosphorylation also modified the Mg2+ requirements and the pattern of inhibition by rolipram, Cyanogen bromide cleavage of the P-32-labeled rat PDE4D3 yielded two or three major phosphopeptide bands, providing a first indication that the enzyme may be phosphorylated at multiple sites in a cell free system, Site-directed mutagenesis was performed on the serine residues present at the amino terminus of this PDE in the context of preferred motifs for PKA phosphorylation. The PKA-dependent incorporation of P-32 was reduced to the largest extent in mutants with both Ser(13) --> Ala and Ser(54) --> Ala substitutions, confirming the presence of more than one phosphorylation site in rat PDE4D3, While substitution of serine 13 with alanine did not affect the activation by PKA, substitution of Ser(54) completely suppressed the kinase activation, Similar conclusions were reached with wild type and mutated PDE4D3 proteins expressed in MA-10 cells, where the endogenous PKA was activated by dibutyryl cAMP. Again, the PDE with the Ser(54) --> Ala substitution could not be activated by the endogenous PKA in the intact cell, These findings support the hypothesis that the PDE4D3 variant contains a regulatory domain target for phosphorylation at the amino terminus of the protein and that Ser(54) in this domain plays a crucial role in activation.