Abstract
MKK7 is a recently discovered mitogen-activated protein kinase (MAPK) kinase that is unique in that it specifically activates only the c-JUN NH(2)-terminal protein kinase (JNK) family of enzymes. Very little is known about the biological role of MKK7. We generated inducible cell lines from the human embryonal kidney carcinoma cell line, HEK293, by stable transfection with a constitutively active mutant of MKK7, MKK7(3E), fused to green fluorescent protein (GFP), under the control of an ecdysone-inducible promoter. Treatment of cells with the synthetic ecdysone analog ponasterone A induced expression of GFP-MKK7(3E) and resulted in sustained activation of endogenous JNK, but neither of the other endogenous MAPKs, ERK or p38. Red and green fluorescing cDNA copies of mRNA extracted from cells obtained before and after induction of GFP-MKK7(3E) were hybridized to microarrays containing more than 6,000 cDNAs in eight independent experiments. By selection criteria, 23 genes were differentially regulated after 24 h of induction of GFP-MKK7(3E) and 16 after 48 h. The expression of 9 genes was consistently changed after both 24 and 48 h of induction. These changes included down-regulation of three genes, c-myc, angiopoietin-2, and glucose-regulated protein 58, and up-regulation of 6 genes, tissue factor pathway inhibitor-2, GRP78, autotaxin, PPP1R7, the DKFZ cDNA p434D0818, and 1 unknown gene. Consistent with previously described roles of several of the altered genes, MKK7(3E) inhibited cell proliferation. These data implicate active MKK7 in the negative regulation of cell proliferation and provide evidence for a new role for this kinase in the regulation of a distinct, hitherto unrecognized set of genes.
Highlights
From the ‡Institute of Pharmacology, Medical School Hannover, Carl-Neuberg Strasse 1, D-30625 Hannover, Germany, and the ¶Molecular Genetics Section, Laboratory of Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255
Transient transfection experiments revealed that green fluorescent protein (GFP) did not compromise the function of MKK73E compared with a version of the enzyme-tagged NH2terminally with a Myc epitope
Like endogenous MKK7, GFP-MKK73E was expressed in the cytosol and in the nucleus, demonstrating that the epitope tag and the mutations did not alter cellular distribution of GFP-MKK73E (Fig. 1, C and D)
Summary
From the ‡Institute of Pharmacology, Medical School Hannover, Carl-Neuberg Strasse 1, D-30625 Hannover, Germany, and the ¶Molecular Genetics Section, Laboratory of Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255. Protein interaction domains on JNKs [17] and on their substrates [18] provide mechanisms to recruit JNK to upstream activators as well as to downstream substrates, such as the transcription factor AP-1 [1, 2] These findings suggest that the specific signaling complexes that are assembled in a particular biological context determine the outcome of the activation of the JNK pathway in each situation [1, 2]. Consistent with this notion, genetic evidence for Drosophila MKK4 and 7, D-MKK4 and Hep/D-MKK-7, respectively, demonstrated that both JNK activators serve distinct and nonredundant functions in flies [1]. Biochemical evidence clearly indicates that MKK7 is an important component of the
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
