Hsp90/p50cdc37 is required for mixed-lineage kinase (MLK) 3 signaling.
Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase (MAPK) kinase kinase that activates MAPK pathways, including the c-Jun NH(2)-terminal kinase (JNK) and p38 pathways. MLK3 and its family members have been implicated in JNK-mediated apoptosis. A survey of human cell lines revealed high levels of MLK3 in breast cancer cells. To learn more about MLK3 regulation and its signaling pathways in breast cancer cells, we engineered the estrogen-responsive human breast cancer cell line, MCF-7, to stably, inducibly express FLAG epitope-tagged MLK3. FLAG.MLK3 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by liquid chromatography/tandem mass spectrometry. Among the proteins identified were heat shock protein 90alpha,beta (Hsp90) and its kinase-specific co-chaperone p50(cdc37). We show that endogenous MLK3 complexes with Hsp90 and p50(cdc37). Further experiments demonstrate that MLK3 associates with Hsp90/p50(cdc37) through its catalytic domain in an activity-independent manner. Upon treatment of MCF-7 cells with geldanamycin, an ansamycin antibiotic that inhibits Hsp90 function, MLK3 levels decrease dramatically. Furthermore, tumor necrosis factor alpha-induced activation of MLK3 and JNK in MCF-7 cells is blocked by geldanamycin treatment. Our finding that geldanamycin treatment does not affect the cellular levels of the downstream signaling components, MAPK kinase 4, MAPK kinase 7, and JNK, suggests that Hsp90/p50(cdc37) regulates JNK signaling at the MAPK kinase kinase level. Previously identified Hsp90/p50(cdc37) clients include oncoprotein kinases and protein kinases that promote cellular proliferation and survival. Our findings reveal that Hsp90/p50(cdc37) also regulates protein kinases involved in apoptotic signaling.
Cell and Molecular Biology Program, Department of Physiology, Michigan State University, 4180 Biomedical and Physical Sciences Building, East Lansing, MI 48824, USA.
Hua
H
Zhang
Wei
W
Wu
Yan
Y
Du
Sarah J
SJ
Santos
Susan E
SE
Conrad
Jack T
JT
Watson
Nicholas
N
Grammatikakis
Kathleen A
KA
Gallo
D017209:Apoptosis; D016227:Benzoquinones; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002455:Cell Division; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D018833:Chaperonins; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins / Q000378:metabolism / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D047029:Lactams, Macrocyclic; D048670:MAP Kinase Kinase 4; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D013058:Mass Spectrometry; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011809:Quinones / Q000494:pharmacology; D015398:Signal Transduction; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; D000099151:MAP Kinase Kinase Kinase 7
D016227:Benzoquinones; D018797:Cell Cycle Proteins; D018833:Chaperonins; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D005982:Glutathione Transferase; D018841:HSP90 Heat-Shock Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D047029:Lactams, Macrocyclic; D048670:MAP Kinase Kinase 4; D020930:MAP Kinase Kinase Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018832:Molecular Chaperones; D011809:Quinones; D014357:Trypsin; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; D000099151:MAP Kinase Kinase Kinase 7; C498362:CDC37 protein, human; C482559:MAP2K4 protein, human; C001277:geldanamycin