Cha H, Lee E K, Shapiro P
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, USA.
J Biol Chem. 2001 Dec 21;276(51):48494-501. doi: 10.1074/jbc.M107601200. Epub 2001 Oct 16.
The C-terminal region of mitogen-activated protein kinase kinase-1 and 2 (MKK1 and MKK2) may function in regulating interactions with upstream kinases or the magnitude and duration of ERK mitogen-activated protein kinase activity. The MKK C-terminal region contains a proline-rich region that reportedly functions in regulating interactions with the Raf-1 kinase and ERK activity. In addition, phosphorylation sites in the C terminus of MKK1 have been suggested to either sustain or attenuate MKK1 activity. To further understand how phosphorylation at the C terminus of MKK1 and protein interactions regulate MKK1 function, we have generated several MKK1 C-terminal deletion mutants and examined their function in regulating MKK1 localization, ERK protein activation, and cell growth. A deletion of C-terminal amino acids encompassing two putative alpha-helices between residues 330 and 379 caused a re-distribution of mutant MKK1 proteins to membrane compartments. Immunofluorescence analysis of MKK1 mutants revealed a loss of homogenous cytosolic distribution that is typically observed with MKK1 wild type, suggesting this region regulates MKK1 cellular localization. In contrast, MKK1 C-terminal deletion mutants localized to various sized punctate regions that overlapped with lysosome compartments. ERK activation in response to constitutively active Raf-1 or growth factor stimulus was attenuated in cells expressing MKK1 C-terminal deletion mutants. This could be partly explained by the inability of Raf-1 to phosphorylate MKK1 C-terminal deletion mutants even though the phosphorylation sites were intact in these mutants. Finally, we show that cells expressing MKK1 C-terminal deletion mutants displayed characteristic patterns of apoptotic cell death and reduced cell proliferation. These findings identify a novel C-terminal region between amino acid residues 330 and 379 on MKK1 that is necessary for regulating the cytoplasmic distribution and subsequent ERK protein activation necessary for cell survival and viability.
丝裂原活化蛋白激酶激酶1和2（MKK1和MKK2）的C末端区域可能在调节与上游激酶的相互作用或细胞外信号调节激酶（ERK）丝裂原活化蛋白激酶活性的大小和持续时间中发挥作用。MKK的C末端区域包含一个富含脯氨酸的区域，据报道该区域在调节与Raf-1激酶的相互作用和ERK活性中发挥作用。此外，有人提出MKK1 C末端的磷酸化位点可维持或减弱MKK1活性。为了进一步了解MKK1 C末端的磷酸化和蛋白质相互作用如何调节MKK1功能，我们构建了几个MKK1 C末端缺失突变体，并检测了它们在调节MKK1定位、ERK蛋白激活和细胞生长方面的功能。缺失330至379位残基之间包含两个假定α螺旋的C末端氨基酸，导致突变型MKK1蛋白重新分布到膜区室。对MKK1突变体的免疫荧光分析显示，失去了野生型MKK1通常具有的均匀胞质分布，表明该区域调节MKK1的细胞定位。相反，MKK1 C末端缺失突变体定位于与溶酶体区室重叠的各种大小的点状区域。在表达MKK1 C末端缺失突变体的细胞中，对组成型活性Raf-1或生长因子刺激的ERK激活减弱。这可以部分解释为，尽管这些突变体中的磷酸化位点完整，但Raf-1无法磷酸化MKK1 C末端缺失突变体。最后，我们表明，表达MKK1 C末端缺失突变体的细胞表现出凋亡性细胞死亡的特征模式，细胞增殖减少。这些发现确定了MKK1上330至379位氨基酸残基之间的一个新的C末端区域，该区域对于调节细胞存活和活力所必需的细胞质分布以及随后的ERK蛋白激活是必需的。
