Katoh Yoshiko, Takemori Hiroshi, Min Li, Muraoka Masaaki, Doi Junko, Horike Nanao, Okamoto Mitsuhiro
Department of Biochemistry and Molecular Biology, Graduate School of Medicine (H-1), Osaka University, Japan.
Eur J Biochem. 2004 Nov;271(21):4307-19. doi: 10.1111/j.1432-1033.2004.04372.x.
Salt-inducible kinase-1 (SIK1) is phosphorylated at Ser577 by protein kinase A in adrenocorticotropic hormone-stimulated Y1 cells, and the phospho-SIK1 translocates from the nucleus to the cytoplasm. The phospho-SIK1 is dephosphorylated in the cytoplasm and re-enters the nucleus several hours later. By using green-fluorescent protein-tagged SIK1 fragments, we found that a peptide region (586-612) was responsible for the nuclear localization of SIK1. The region was named the 'RK-rich region' because of its Arg- and Lys-rich nature. SIK1s mutated in the RK-rich region were localized mainly in the cytoplasm. Because SIK1 represses cAMP-response element (CRE)-mediated transcription of steroidogenic genes, the mutants were examined for their effect on transcription. To our surprise, the cytoplasmic mutants strongly repressed the CRE-binding protein (CREB) activity, the extent of repression being similar to that of SIK1(S577A), a mutant localized exclusively in the nucleus. Several chimeras were constructed from SIK1 and from its isoform SIK2, which was localized mainly in the cytoplasm, and they were examined for intracellular localization as well as CREB-repression activity. A SIK1-derived chimera, where the RK-rich region had been replaced with the corresponding region of SIK2, was found in the cytoplasm, its CREB-modulating activity being similar to that of wild-type SIK1. On the other hand, a SIK2-derived chimera with the RK-rich region of SIK1 was localized in both the nucleus and the cytoplasm, and had a CREB-repressing activity similar to that of the wild-type SIK2. Green fluorescent protein-fused transducer of regulated CREB activity 2 (TORC2), a CREB-specific co-activator, was localized in the cytoplasm and nucleus of Y1 cells, and, after treatment with adrenocorticotropic hormone, cytoplasmic TORC2 entered the nucleus, activating CREB. The SIK1 mutants, having a strong CRE-repressing activity, completely inhibited the adrenocorticotropic hormone-induced nuclear entry of green fluorescent protein-fused TORC2. This suggests that SIK1 may regulate the intracellular movement of TORC2, and as a result modulates the CREB-dependent transcription activity. Together, these results indicate that the RK-rich region of SIK1 is important for determining the nuclear localization and attenuating CREB-repressing activity, but the degree of the nuclear localization of SIK1 itself does not necessarily reflect the degree of SIK1-mediated CREB repression.
在促肾上腺皮质激素刺激的Y1细胞中，盐诱导激酶1（SIK1）在丝氨酸577位点被蛋白激酶A磷酸化，磷酸化的SIK1从细胞核转运至细胞质。磷酸化的SIK1在细胞质中去磷酸化，并在数小时后重新进入细胞核。通过使用绿色荧光蛋白标记的SIK1片段，我们发现一个肽段区域（586 - 612）负责SIK1的核定位。由于该区域富含精氨酸和赖氨酸，故被命名为“富含RK区域”。在富含RK区域发生突变的SIK1主要定位于细胞质中。因为SIK1抑制环磷酸腺苷反应元件（CRE）介导的类固醇生成基因转录，所以对这些突变体的转录作用进行了检测。令我们惊讶的是，细胞质突变体强烈抑制CRE结合蛋白（CREB）的活性，其抑制程度与仅定位于细胞核中的突变体SIK1（S577A）相似。由SIK1及其主要定位于细胞质中的同工型SIK2构建了几个嵌合体，并对它们的细胞内定位以及CREB抑制活性进行了检测。一个源自SIK1的嵌合体，其富含RK区域被SIK2的相应区域取代，该嵌合体定位于细胞质中，其对CREB的调节活性与野生型SIK1相似。另一方面，一个带有SIK1富含RK区域的源自SIK2的嵌合体定位于细胞核和细胞质中，并且具有与野生型SIK2相似的CREB抑制活性。绿色荧光蛋白融合的调节CREB活性转导子2（TORC2），一种CREB特异性共激活因子，定位于Y1细胞的细胞质和细胞核中，在用促肾上腺皮质激素处理后，细胞质中的TORC2进入细胞核，激活CREB。具有强大CRE抑制活性的SIK1突变体完全抑制了促肾上腺皮质激素诱导的绿色荧光蛋白融合的TORC2进入细胞核。这表明SIK1可能调节TORC2的细胞内转运，从而调节CREB依赖的转录活性。总之，这些结果表明，SIK1的富含RK区域对于确定核定位和减弱CREB抑制活性很重要，但SIK1本身的核定位程度不一定反映SIK1介导的CREB抑制程度。
