Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus.
Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus.
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DOI：
10.1083/jcb.126.4.839
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发表时间：
1994-08
期刊：
影响因子：
--
通讯作者：
Schulman H
中科院分区：
文献类型：
--
作者：
Srinivasan M;Edman CF;Schulman H
细胞内靶向可以使具有广泛底物特异性的蛋白激酶，如多功能Ca 2 +/钙调蛋白依赖性蛋白激酶（CaM激酶）能够实现体内作用的选择性。我们已经研究了三个delta-CaM激酶亚型的细胞内靶向。δ B-CaM激酶同种型靶向转染细胞的细胞核，而δ A-和δ C-CaM激酶同种型是细胞溶质/细胞骨架。含有δ B-CaM激酶可变结构域的α-CaM激酶的嵌合构建体被重新路由至细胞核，而天然α-CaM激酶和含有δ A-或δ C-CaM激酶可变结构域的α-CaM激酶的嵌合体被保留在细胞质中。使用定点诱变，我们已经定义了一个核定位信号（NLS）内的11个氨基酸的序列，可能通过选择性剪接插入，在可变域的δ B-CaM激酶。CaM激酶的亚型特异性核靶向可能是CaM激酶选择性调节核功能的关键机制。CaM激酶是一种多聚体，可由几种亚型组成。我们发现，当细胞表达两种不同的钙调素激酶亚型时，细胞靶向由亚型的比例决定。当过量的CaM激酶的细胞质亚型沿着细胞核亚型共表达时，两种亚型都定位于细胞质中。相反，过量的核同种型可以将细胞质同种型重新路由到细胞核。核同种型可能与胞质同种型共组装，以形成被转运到核中的异源多聚体全酶。这些实验证明了CaM激酶的同种型特异性靶向，并表明这种靶向可以通过表达该酶的多种同种型来修饰。
Intracellular targeting may enable protein kinases with broad substrate- specificities, such as multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) to achieve a selectivity of action in vivo. We have examined the intracellular targeting of three delta-CaM kinase isoforms. The delta B-CaM kinase isoform is targeted to the nucleus in transfected cells while the delta A- and delta C-CaM kinase isoforms are cytosolic/cytoskeletal. A chimeric construct of alpha-CaM kinase containing the delta B-CaM kinase variable domain is rerouted to the nucleus while the native alpha-CaM kinase and chimeras of alpha-CaM kinase which contain the delta A- or delta C-CaM kinase variable domains are retained in the cytoplasm. Using site-directed mutagenesis, we have defined a nuclear localization signal (NLS) within an 11-amino acid sequence, likely inserted by alternative splicing, in the variable domain of delta B-CaM kinase. Isoform-specific nuclear targeting of CaM kinase is probably a key mechanism in the selective regulation of nuclear functions by CaM kinase. CaM kinase is a multimer that can be composed of several isoforms. We find that when cells express two different isoforms of CaM kinase, cellular targeting is determined by the ratio of the isoforms. When an excess of the cytoplasmic isoform of CaM kinase is coexpressed along with the nuclear isoform, both isoforms are localized in the cytoplasm. Conversely an excess of the nuclear isoform can reroute the cytoplasmic isoform to the nucleus. The nuclear isoform likely coassembles with the cytosolic isoform, to form a heteromultimeric holoenzyme which is transported into the nucleus. These experiments demonstrate isoform-specific targeting of CaM kinase and indicate that such targeting can be modified by the expression of multiple isoforms of the enzyme.