Identification of a C-terminal region that regulates mitogen-activated protein kinase kinase-1 cytoplasmic localization and ERK activation.
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.
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Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, USA.
Lee
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Shapiro
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D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D002470:Cell Survival; D003593:Cytoplasm / Q000201:enzymology*; D017931:DNA Primers; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D017931:DNA Primers; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases