Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Biochem J. 2010 Aug 15;430(1):39-48. doi: 10.1042/BJ20091758.
The HMG (high-mobility group)-box-containing chromatin-remodelling factor SRY (sex-determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus is critically dependent on two NLSs (nuclear localization signals) that flank its HMG domain: the C-terminally located 'beta-NLS' that mediates nuclear transport through Impbeta1 (importin beta1) and the N-terminally located 'CaM-NLS' which is known to recognize the calcium-binding protein CaM (calmodulin). In the present study, we examined a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localization of GFP (green fluorescent protein)-SRY fusion proteins in transfected cells compared with wild-type. The CaM antagonist CDZ (calmidazolium chloride) was found to significantly reduce wild-type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ's effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM-binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG-box-domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found for the first time to rely on CaM for optimal nuclear localization. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors.
高迁移率族盒结构域染色质重塑因子 SRY（Y 染色体性别决定区）在性别决定中发挥关键作用。其在核内的作用严重依赖于其 HMG 结构域侧翼的两个核定位信号（NLS）：位于 C 端的“β-NLS”，通过 Impbeta1（importin beta1）介导核运输，以及位于 N 端的“CaM-NLS”，已知其识别钙结合蛋白 CaM（钙调蛋白）。在本研究中，我们首次检查了来自人类 XY 性反转女性的 SRY CaM-NLS 的多种错义突变，结果表明它们导致转染细胞中 GFP（绿色荧光蛋白）-SRY 融合蛋白的核定位显著减少，与野生型相比。发现 CaM 拮抗剂 CDZ（calmidazolium chloride）可显著减少野生型 SRY 的核积累，表明 SRY 核输入依赖于 CaM。有趣的是，CaM-NLS 突变体均对 CDZ 的作用具有抗性，暗示与 CaM 的相互作用丧失，这通过直接结合实验得到证实。CaM 结合/导致核积累是唯一发现受 CaM-NLS 两种突变影响的 SRY 特性，这表明抑制 CaM 依赖性核输入是这些情况下性别反转的基础。重要的是，CaM-NLS 在其他 HMG 盒结构域包含蛋白中是保守的，如 SOX-2、-9、-10 和 HMGN1，它们都首次被发现依赖于 CaM 进行最佳核定位。因此，CaM 依赖性核易位是这一家族重要转录因子的共同机制。
