title: Interferon-gamma-dependent tyrosine phosphorylation of MEKK4 via Pyk2 is regulated by annexin II and SHP2 in keratinocytes.
abstract: IFNgamma (interferon-gamma) binding to its cognate receptor results, through JAK (Janus kinase), in direct activation of receptor-bound STAT1 (signal transducer and activator of transcription 1), although there is evidence for additional activation of a MAPK (mitogen-activated protein kinase) pathway. In the present paper, we report IFNgamma-dependent activation of the MEKK4 (MAPK/extracellular-signal-regulated kinase kinase kinase 4) pathway in HaCaT human keratinocytes. MEKK4 is tyrosine-phosphorylated and the IFNgamma-dependent phosphorylation requires intracellular calcium. Calcium-dependent phosphorylation of MEKK4 is mediated by Pyk2. Moreover, MEKK4 and Pyk2 co-localize in an IFNgamma-dependent manner in the perinuclear region. Furthermore, the calcium-binding protein, annexin II, and the calcium-regulated kinase, Pyk2, co-immunoprecipitate with MEKK4 after treatment with IFNgamma. Immunofluorescence imaging of HaCaT cells shows an IFNgamma-dependent co-localization of annexin II with Pyk2 in the perinuclear region, suggesting that annexin II mediates the calcium-dependent regulation of Pyk2. Tyrosine phosphorylation of MEKK4 correlates with its activity to phosphorylate MKK6 (MAPK kinase 6) in vitro and subsequent p38 MAPK activation in an IFNgamma-dependent manner. Additional studies demonstrate that the SH2 (Src homology 2)-domain-containing tyrosine phosphatase SHP2 co-immunoprecipitates with MEKK4 in an IFNgamma-dependent manner and co-localizes with MEKK4 after IFNgamma stimulation in the perinuclear region in HaCaT cells. Furthermore, we provide evidence that SHP2 dephosphorylates MEKK4 and Pyk2, terminating the MEKK4-dependent branch of the IFNgamma signalling pathway.
authors.affiliation: Department of Pharmacology and Toxicology, University of Arizona, 1703 E. Mabel Street, Tucson, AZ 85721, USA.
authors.forename: Ursula M
authors.initials: UM
authors.lastname: Halfter
authors.forename: Zachary E
authors.initials: ZE
authors.lastname: Derbyshire
authors.forename: Richard R
authors.initials: RR
authors.lastname: Vaillancourt
mesh_terms: D000818:Animals; D017306:Annexin A2 / Q000737:chemistry / Q000502:physiology*; D020013:Calcium Signaling; D002460:Cell Line; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000502:physiology*; D015603:Keratinocytes / Q000502:physiology; D048669:MAP Kinase Kinase 6 / Q000502:physiology; D048768:MAP Kinase Kinase Kinase 4 / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine
chemical_list: D017306:Annexin A2; D014443:Tyrosine; D007371:Interferon-gamma; D048768:MAP Kinase Kinase Kinase 4; D048669:MAP Kinase Kinase 6; C482566:MAP2K6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11