14-3-3 is phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in vivo regulates Raf/14-3-3 interaction.
14-3-3 proteins mediate interactions between proteins involved in signal transduction and cell cycle regulation. Phosphorylation of target proteins as well as 14-3-3 are important for protein-protein interactions. Here, we describe the purification of a protein kinase from porcine brain that phosphorylates 14-3-3 zeta on Thr-233. This protein kinase has been identified as casein kinase Ialpha (CKIalpha) by peptide mapping analysis and sequencing. Among mammalian 14-3-3, only 14-3-3 tau possesses a phosphorylatable residue at the same position (Ser-233), and we show that this residue is also phosphorylated by CKI. In addition, we show that 14-3-3 zeta is exclusively phosphorylated on Thr-233 in human embryonic kidney 293 cells. The residue 233 is located within a region shown to be important for the association of 14-3-3 to target proteins. We showed previously that, in 293 cells, only the unphosphorylated form of 14-3-3 zeta associates with the regulatory domain of c-Raf. We have now shown that in vivo phosphorylation of 14-3-3 zeta at the CKIalpha site (Thr-233) negatively regulates its binding to c-Raf, and may be important in Raf-mediated signal transduction.
Dubois
T
T
Division of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom. t-dubois@nimr.mrc.ac.uk
Rommel
C
C
Howell
S
S
Steinhussen
U
U
Soneji
Y
Y
Morrice
N
N
Moelling
K
K
Aitken
A
A
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D047388:Casein Kinases; D002461:Cell Line, Transformed; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013552:Swine; D013912:Threonine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D011506:Proteins; D011994:Recombinant Proteins; D013912:Threonine; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D047388:Casein Kinases; D019908:Proto-Oncogene Proteins c-raf