Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein.
Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.
Axxima Pharmaceuticals AG, 82152 Martinsried, Germany. daub@axxima.com
Henrik
H
Daub
Stephanie
S
Blencke
Peter
P
Habenberger
Alexander
A
Kurtenbach
Julia
J
Dennenmoser
Josef
J
Wissing
Axel
A
Ullrich
Matt
M
Cotten
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D019556:COS Cells; D002460:Cell Line; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D006509:Hepatitis B / Q000188:drug therapy / Q000201:enzymology / Q000821:virology; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D006512:Hepatitis B Core Antigens; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C087728:SRPK1 protein, human; D017346:Protein Serine-Threonine Kinases; C110522:SRPK2 protein, human; D011493:Protein Kinase C; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases