Genomic tagging reveals a random association of endogenous PtdIns5P 4-kinases IIalpha and IIbeta and a partial nuclear localization of the IIalpha isoform.
Genomic tagging reveals a random association of endogenous PtdIns5P 4-kinases IIalpha and IIbeta and a partial nuclear localization of the IIalpha isoform.
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DOI：
10.1042/bj20100340
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发表时间：
2010-09-01
期刊：
影响因子：
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通讯作者：
Clarke JH
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文献类型：
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作者：
Wang M;Bond NJ;Letcher AJ;Richardson JP;Lilley KS;Irvine RF;Clarke JH
ptdins5p4激酶i α和i β转染到包括DT40细胞在内的细胞后分别为胞质和细胞核[Richardson， Wang, Clarke， Patel和Irvine (2007) Cell]。信号处理[j]。在目前的研究中，我们在DT40细胞中对两种II型ptdins5p4 -激酶亚型进行了基因组标记。从标记细胞中免疫沉淀两种异构体，然后进行MS，显示它们彼此直接相关，可能是通过异源二聚化。我们用实时定量PCR定量了II型ptdins5p4 -激酶mrna的细胞水平，用14N、13c标记的内标准肽选择性反应监测，用质谱法定量了免疫沉淀物中每个亚型的绝对数量。结果表明，二聚化是完全和随机的，完全由两种同工异构体的相对浓度决定。正如预期的那样，ptdins4p4 -激酶IIβ是bb0 95%的核，而ptdins4p4 -激酶IIα的分布是60%的细胞质（都与膜结合）和40%的核。在体外，ptdins5p4激酶i α的活性是IIβ亚型的2000倍。总的来说，结果表明ptdins5p4 -激酶ii - β的功能可能是将更活跃的ii - α亚型靶向到细胞核中。
PtdIns5P 4-kinases IIα and IIβ are cytosolic and nuclear respectively when transfected into cells, including DT40 cells [Richardson, Wang, Clarke, Patel and Irvine (2007) Cell. Signalling 19, 1309–1314]. In the present study we have genomically tagged both type II PtdIns5P 4-kinase isoforms in DT40 cells. Immunoprecipitation of either isoform from tagged cells, followed by MS, revealed that they are associated directly with each other, probably by heterodimerization. We quantified the cellular levels of the type II PtdIns5P 4-kinase mRNAs by real-time quantitative PCR and the absolute amount of each isoform in immunoprecipitates by MS using selective reaction monitoring with 14N,13C-labelled internal standard peptides. The results suggest that the dimerization is complete and random, governed solely by the relative concentrations of the two isoforms. Whereas PtdIns5P 4-kinase IIβ is >95% nuclear, as expected, the distribution of PtdIns4P 4-kinase IIα is 60% cytoplasmic (all bound to membranes) and 40% nuclear. In vitro, PtdIns5P 4-kinase IIα was 2000-fold more active as a PtdIns5P 4-kinase than the IIβ isoform. Overall the results suggest a function of PtdIns5P 4-kinase IIβ may be to target the more active IIα isoform into the nucleus.