The identification of phosphorylation sites of pp32 and biochemical purification of a cellular pp32-kinase.
The versatile phosphoprotein pp32 is involved in important physiological processes, including cell proliferation, apoptosis, mRNA transport, and transcription. We have previously reported that pp32, through histone masking, inhibits histone acetylation and transcriptional activation by histone acetyltransferases. However, how pp32 itself is regulated remained largely unknown. Although pp32 is a phosphoprotein, neither the phosphorylation sites nor the cellular kinase has been identified. In this report, utilizing an in vitro kinase assay and a biochemical purification scheme, we identify casein kinase II as a cellular pp32-kinase. Our deletion and site-specific mutagenesis studies identify serines 158 and 204 as the sites of phosphorylation. Generation and utilization of antibodies with higher affinity for phospho-pp32 demonstrate that pp32 is indeed phosphorylated in vivo at these two sites. Mutagenesis studies on pp32 suggest a role for serines 158 and 204 in its function. The identification of the pp32 kinase and the sites of pp32 phosphorylation as well as the generation of antibodies with higher affinity for phospho-pp32 should now provide key information and tools for future studies on pp32 regulation.
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA.
Rui
R
Hong
Todd
T
Macfarlan
Sara N
SN
Kutney
Sang-Beom
SB
Seo
Yuki
Y
Mukai
Felix
F
Yelin
Gary R
GR
Pasternack
Debabrata
D
Chakravarti
D000595:Amino Acid Sequence; D047390:Casein Kinase II; D002467:Cell Nucleus / Q000201:enzymology; D004004:Dichlororibofuranosylbenzimidazole / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D006367:HeLa Cells; D006493:Heparin / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D017384:Sequence Deletion; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014162:Transfection
D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D012694:Serine; D004004:Dichlororibofuranosylbenzimidazole; D006493:Heparin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases