Phosphorylation of serum response factor by casein kinase II: evidence against a role in growth factor regulation of fos expression.
Serum response factor (SRF) is a transcription factor involved in the serum and growth factor regulation of the c-fos gene. SRF is phosphorylated by casein kinase II (CKII), which causes a large increase in its DNA-binding activity. CKII activity has been shown to be stimulated by growth factors and serum. Since c-fos transcription is induced by a number of the same agents that stimulate CKII activity, and since fos activation and CKII stimulation demonstrate similar rapid kinetics, a role was proposed for CKII in regulating fos expression via its phosphorylation of SRF. In this report, we provide evidence against this hypothesis by using several different strategies. First, by immunoprecipitation of SRF from cells, we show that the phosphorylation state of SRF does not change upon growth factor treatment. Second, by two-dimensional electrophoresis of lysates from a cell line that overexpresses SRF, we show that, although SRF exists in the cell in several different isoforms, there is no change in their relative amounts upon serum stimulation. Third, we tested the activity of an SRF mutant that binds DNA at constitutively high levels irrespective of CKII phosphorylation. If phosphorylation is regulatory, this mutant would be expected to constitutively activate (or repress) fos expression. However, when overexpressed stably in cells this mutant had no effect on endogenous c-fos expression, suggesting that CKII phosphorylation of SRF is not the limiting event for fos activation.
Manak
J R
JR
Department of Biological Sciences, Columbia University, New York, New York 10027.
Prywes
R
R
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II; D002918:Chymotrypsin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation; D016762:Genes, fos; D006133:Growth Substances / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D026362:Serum Response Factor; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D004268:DNA-Binding Proteins; D006133:Growth Substances; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D002918:Chymotrypsin; D014357:Trypsin