Plasma membrane-targeted Raf kinase activates NF-kappaB and human immunodeficiency virus type 1 replication in T lymphocytes.
Increasing evidence points to a role of the mitogenic Ras/Raf/MEK/ERK signaling cascade in regulation of human immunodeficiency virus type 1 (HIV-1) gene expression. Stimulation of elements of this pathway leads to transactivation of the HIV-1 promoter. In particular, the NF-kappaB motif in the HIV long terminal repeat (LTR) represents a Raf-responsive element in fibroblasts. Regulation of the Raf kinase in T cells differs from findings with a variety of cell lines that the catalytic domain of Raf (Raf(delta26-303)) shows no activity. In this study, we restored the activity of the kinase in T cells by fusing its catalytic domain to the CAAX motif (-Cx) of Ras, thus targeting the enzyme to the plasma membrane. Constitutive activity of Raf was demonstrated by phosphorylation of mitogen-activated protein kinase kinase (MEK) and endogenous mitogen-activated protein kinase 1/2 (ERK1/2) in A3.01 T cells transfected with Raf(delta26-303)-Cx. Membrane-targeted Raf also stimulates NF-kappaB, as judged by kappaB-dependent reporter assays and enhanced NF-kappaB p65 binding on band shift analysis. Moreover, we found that active Raf transactivates the HIV(NL4-3) LTR in A3.01 T lymphocytes and that dominant negative Raf (C4) blocked 12-O-tetradecanoylphorbol-13-acetate induced transactivation. When cotransfected with infectious HIV(NL4-3) DNA, membrane-targeted Raf induces viral replication up to 10-fold over basal levels, as determined by the release of newly synthesized p24gag protein. Our study clearly demonstrates that the activity of the catalytic domain of Raf in A3.01 T cells is dependent on its cellular localization. The functional consequences of active Raf in T lymphocytes include not only NF-kappaB activation and transactivation of the HIV(NL4-3) LTR but also synthesis and release of HIV particles.
Flory
E
E
Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg, Germany.
Weber
C K
CK
Chen
P
P
Hoffmeyer
A
A
Jassoy
C
C
Rapp
U R
UR
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism / Q000821:virology*; D015533:Transcriptional Activation; D014779:Virus Replication
D016328:NF-kappa B; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human