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Originally published In Press as doi:10.1074/jbc.M107601200 on October 16, 2001
J. Biol. Chem., Vol. 276, Issue 51, 48494-48501, December 21, 2001
Identification of a C-terminal Region That Regulates
Mitogen-activated Protein Kinase Kinase-1 Cytoplasmic Localization
and ERK Activation*
Hyukjin
Cha,
Eun Kyoung
Lee, and
Paul
Shapiro
From the Department of Pharmaceutical Sciences, University of
Maryland School of Pharmacy, Baltimore, Maryland 21201
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
-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.
*
This work was supported by a grant from the Concern
Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 410-706-8522;
Fax: 410-706-0346; E-mail: pshapiro@rx.umaryland.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
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