The nuclear import of protein kinase D3 requires its catalytic activity.
The protein kinase D (PKD) family consists of three serine/threonine protein kinases termed PKD, PKD2, and PKD3, which are similar in overall structure and primary amino acid sequence. However, each isozyme displays a distinctive intracellular localization. Taking advantage of the structural homology and opposite nuclear localization of PKD2 and PKD3, we generated an extensive set of chimeric proteins between both isozymes to determine which PKD3 domain(s) mediates its nuclear localization. We found that the C-terminal region of PKD3, which contains its catalytic domain, is necessary but not sufficient for its nuclear localization. Real time imaging of a photoactivatable green fluorescent protein fused to PKD3 revealed that point mutations that render PKD3 catalytically inactive completely prevented its nuclear import despite its interaction with importin alpha and beta. We also found that activation loop phosphorylation of PKD3 did not require its nuclear localization, and it was not sufficient to promote the nuclear import of PKD3. These results identify a novel function for the kinase activity of PKD3 in promoting its nuclear entry and suggest that the catalytic activity of PKD3 may regulate its nuclear import through autophosphorylation and/or interaction with another protein(s).
Rey
Osvaldo
O
Division of Digestive Diseases, Department of Medicine, CURE Digestive Diseases Research Center and Molecular Biology Institute, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1786, USA. orey@mednet.ucla.edu
Papazyan
Romeo
R
Waldron
Richard T
RT
Young
Steven H
SH
Lippincott-Schwartz
Jennifer
J
Jacamo
Rodrigo
R
Rozengurt
Enrique
E
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D028961:beta Karyopherins / Q000378:metabolism
D009841:Oligonucleotides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D028961:beta Karyopherins; C119190:protein kinase C nu; D011493:Protein Kinase C