Phospho-specific binding of 14-3-3 proteins to phosphatidylinositol 4-kinase III β protects from dephosphorylation and stabilizes lipid kinase activity
Phospho-specific binding of 14-3-3 proteins to phosphatidylinositol 4-kinase III β protects from dephosphorylation and stabilizes lipid kinase activity
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DOI：
10.1242/jcs.03104
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发表时间：
2006-09-01
影响因子：
4
通讯作者：
Pfizenmaier, Klaus
中科院分区：
文献类型：
--
作者：
Hausser, Angelika;Link, Gisela;Pfizenmaier, Klaus
磷脂酰肌醇-4-激酶-III β（PI 4KIII β）通过PKD介导的磷酸化在高尔基体区室被激活。随后负责在高尔基体膜上持续产生PtdIns（4）P的机制和活化的PI 4KIII β的潜在相互作用伴侣尚不清楚。在这里，我们确定磷酸丝氨酸/-苏氨酸结合14-3-3蛋白作为这种磷酸化下游的PI 4KIII β活性的新调节剂。PI 4KIII β-14-3-3相互作用，从GST下拉，免疫共沉淀和双分子荧光互补中明显可见，通过用冈田酸抑制磷酸酶而增强。14-3-3蛋白与PI 4KIII β的结合涉及PKD磷酸化位点Ser 294，这从14-3-3与S294 A PI 4KIII β突变体的结合减少中可以看出。显性负性14-3-3蛋白的表达导致PI 4KIII β Ser 294磷酸化降低，而野生型14-3-3蛋白增加磷酸化PI 4KIII β水平。这是因为PI 4 KIII β Ser 294磷酸化受到磷酸酶介导的去磷酸化的保护。PI 4KIII β-14-3-3相互作用的功能意义从显性负性143- 3蛋白表达后PI 4KIII β活性的降低是明显的。我们认为，14-3-3蛋白作为PI 4KIII β活性的正调节剂，通过保护脂质激酶免于活性位点去磷酸化，从而确保高尔基体区室持续供应PtdIns（4）P。
Phosphatidylinositol-4-kinase-III beta (PI4KIII beta) is activated at the Golgi compartment by PKD-mediated phosphorylation. Subsequent mechanisms responsible for continuous PtdIns(4)P production at Golgi membranes and potential interaction partners of activated PI4KIII beta are unknown. Here we identify phosphoserine/-threonine binding 14-3-3 proteins as novel regulators of PI4KIII beta activity downstream of this phosphorylation. The PI4KIII beta-14-3-3 interaction, evident from GST pulldowns, co-immunoprecipitations and bimolecular fluorescence complementation, was augmented by phosphatase inhibition with okadaic acid. Binding of 14-3-3 proteins to PI4KIII beta involved the PKD phosphorylation site Ser294, evident from reduced 14-3-3 binding to a S294A PI4KIII beta mutant. Expression of dominant negative 14-3-3 proteins resulted in decreased PI4KIII beta Ser294 phosphorylation, whereas wildtype 14-3-3 proteins increased phospho PI4KIII beta levels. This was because of protection of PI4KIII beta Ser294 phosphorylation from phosphatase-mediated dephosphorylation. The functional significance of the PI4KIII beta-14-3-3 interaction was evident from a reduction of PI4KIII beta activity upon dominant negative 143- 3 protein expression. We propose that 14-3-3 proteins function as positive regulators of PI4KIII beta activity by protecting the lipid kinase from active site dephosphorylation, thereby ensuring a continuous supply of PtdIns(4)P at the Golgi compartment.