The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site.
Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signals trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr(24), Ser(256) and Ser(319)) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser(329)) as a novel in vivo phosphorylation site on FKHR. Ser(329) phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser(329) is phosphorylated in unstimulated HEK-293 cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser(329) specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser(329) in vivo.
Woods
Y L
YL
MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. y.l.woods@dundee.ac.uk
Rena
G
G
Morrice
N
N
Barthel
A
A
Becker
W
W
Guo
S
S
Unterman
T G
TG
Cohen
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015533:Transcriptional Activation; D000097603:Dyrk Kinases
D004268:DNA-Binding Proteins; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D012694:Serine; D014157:Transcription Factors; D000097603:Dyrk Kinases; C085126:FOXO1 protein, human