Nucleoporin phosphorylation triggered by the encephalomyocarditis virus leader protein is mediated by mitogen-activated protein kinases.
Cardioviruses disrupt nucleocytoplasmic transport through the activity of their leader (L) protein. We have shown that hyperphosphorylation of nuclear pore proteins (nucleoporins or Nups), including Nup62, Nup153, and Nup214, is central to this L protein function and requires one or more cytosolic kinases. In this study, potential cellular enzymes involved in encephalomyocarditis virus (EMCV) L-directed Nup phosphorylation were screened with a panel of specific, cell-permeating kinase inhibitors. Extracellular signal-regulated receptor kinase (ERK) and p38 mitogen-activated protein kinase inhibitors (U0126 and SB203580) were sufficient to block Nup hyperphosphorylation in EMCV-infected or L-expressing cells. Recombinant L alone, in the absence of infection, triggered activation of ERK and p38, independent of their upstream signaling cascades. Conserved residues within the L zinc finger (Cys(19)) and acidic domain (Asp(48),(51),(52),(55)) were essential for this activation and for the phosphorylation of Nups, suggesting that the phenomena are linked. Analysis of the hyperphosphorylated Nup species revealed only phosphoserine and phosphothreonine residues. The sizes of the tryptic phosphopeptides derived from Nup62 were compatible with sites in the Phe/Gly repeat domain which display common consensus sequences for ERK and p38 substrates. The results provide strong evidence that ERK and p38 are the probable effector kinases required for L-dependent inhibition of nuclear trafficking.
Porter
Frederick W
FW
Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Brown
Bradley
B
Palmenberg
Ann C
AC
D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D018188:Cardiovirus Infections / Q000235:genetics / Q000378:metabolism / Q000821:virology; D004686:Encephalomyocarditis virus / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis; D011725:Pyridines / Q000494:pharmacology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D002070:Butadienes; D004791:Enzyme Inhibitors; D007093:Imidazoles; D009570:Nitriles; D028861:Nuclear Pore Complex Proteins; D010748:Phosphopeptides; D011725:Pyridines; C113580:U 0126; D014764:Viral Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580