title: The E3 ligase CHIP mediates ubiquitination and degradation of mixed-lineage kinase 3.
abstract: Mixed-lineage kinase 3 (MLK3) activates mitogen-activated protein kinase (MAPK) signaling pathways and has important functions in migration, invasion, proliferation, tumorigenesis, and apoptosis. We investigated the role of the E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) in the regulation of MLK3 protein levels. We show that CHIP interacts with MLK3 and, together with the E2 ubiquitin-conjugating enzyme UbcH5 (UbcH5a, -b, -c, or -d), ubiquitinates MLK3 in vitro. CHIP or Hsp70 overexpression promoted endogenous MLK3 ubiquitination and induced a decline in MLK3 protein levels in cells with Hsp90 inhibition. Furthermore, CHIP overexpression caused a proteasome-dependent reduction in exogenous MLK3 protein. Geldanamycin (GA), heat shock, and osmotic shock treatments also reduced the level of MLK3 protein via a CHIP-dependent mechanism. In addition, CHIP depletion in ovarian cancer SKOV3 cells increased cell invasion, and the enhancement of invasiveness was abrogated by small interfering RNA (siRNA)-mediated knockdown of MLK3. Thus, CHIP modulates MLK3 protein levels in response to GA and stress stimuli, and CHIP-dependent regulation of MLK3 is required for suppression of SKOV3 ovarian cancer cell invasion.
authors.affiliation: Department of Biological Sciences, The University of Toledo, Toledo, Ohio, USA.
authors.forename: Natalya A
authors.initials: NA
authors.lastname: Blessing
authors.affiliation: Department of Biological Sciences, The University of Toledo, Toledo, Ohio, USA.
authors.forename: April L
authors.initials: AL
authors.lastname: Brockman
authors.affiliation: Department of Biological Sciences, The University of Toledo, Toledo, Ohio, USA deborah.chadee@utoledo.edu.
authors.forename: Deborah N
authors.initials: DN
authors.lastname: Chadee
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D018841:HSP90 Heat-Shock Proteins / Q000096:biosynthesis / Q000378:metabolism; D018869:Heat-Shock Response; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D020930:MAP Kinase Kinase Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D009361:Neoplasm Invasiveness; D009997:Osmotic Pressure; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering; D013012:Sorbitol / Q000494:pharmacology; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000235:genetics*; D054875:Ubiquitination; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D020930:MAP Kinase Kinase Kinases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013012:Sorbitol; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C097308:UBE2D1 protein, human; C097309:UBE2D2 protein, human; C097310:UBE2D3 protein, human; C467728:STUB1 protein, human; C001277:geldanamycin