Membrane binding and enzymatic activation of a Dbl homology domain require the neighboring pleckstrin homology domain.
Dbl-homology (DH) domains are invariably located immediately N-terminal to a pleckstrin homology (PH) domain. To understand the functional relationship between these two domains we expressed the DH domain alone, the PH domain alone, and the DH-PH combination of the invasion inducing protein Tiam-1 fused to glutathione-S-transferase (GST) or green fluorescent protein (GFP). We found that the GST-DH-PH and the GST-PH constructs bind to preparations of brain membranes and to the beta gamma subunits of trimeric G proteins in vitro, while the GST-DH and GST control do not. The GFP-DH-PH and GFP-PH constructs are localized to peripheral membranes of COS-7 cells in vivo, while GFP and GFP-DH domain constructs are found diffusely in the cytoplasm. The DH-PH domain combination activates Jun N-terminal kinase (JNK) strongly, but the DH domain alone and the PH domain alone have little effect. We conclude that membrane localization and enzymatic activation of the DH domain require the adjacent PH domain.
Wang
D S
DS
Department of Neuroscience, University of Florida College of Medicine, Gainesville 32610, USA.
Deng
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T
Shaw
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D000818:Animals; D015153:Blotting, Western; D001921:Brain; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D029721:Drosophila Proteins; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D008164:Luminescent Proteins / Q000235:genetics; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D009698:Nucleoproteins / Q000737:chemistry* / Q000235:genetics; D063150:Polycomb Repressive Complex 1; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D008164:Luminescent Proteins; D009698:Nucleoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; C072820:ph-d protein, Drosophila; D049452:Green Fluorescent Proteins; D063150:Polycomb Repressive Complex 1; D005982:Glutathione Transferase; D011494:Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D019204:GTP-Binding Proteins