Demonstration of the phosphorylation-dependent interaction of tryptophan hydroxylase with the 14-3-3 protein.
The molecular mechanism of the phosphorylation-dependent activation of tryptophan hydroxylase is studied with respect to the role of the 14-3-3 protein. Reexamination of the system reconstituted with the purified TRH and the 14-3-3 protein showed that the level of the TRH activity correlated with the extent of the Ca2+/calmodulin- or the cAMP-dependent phosphorylation in TRH. The experiment confirmed the requirement of the 14-3-3 protein for the activation, but the 14-3-3 protein added into the assay mixture did not affect either the extent nor the specificity of the phosphorylation. However, the analysis of the assay mixture on a pteridine-based affinity column indicated the formation of a complex between TRH and the 14-3-3 protein, where the complex formation depended on the phosphorylation of TRH. The complex between the phosphorylated TRH and the 14-3-3 protein could also be detected by analysis of crude brainstem extract previously phosphorylated by endogeneous Ca2+/calmodulin-dependent protein kinase. The 14-3-3 protein, therefore, appears to be a phosphorylation-dependent TRH-binding protein whose interaction causes the activation of TRH.
Furukawa
Y
Y
Department of Biochemistry, Faculty of Science, Niigata University, Japan.
Ikuta
N
N
Omata
S
S
Yamauchi
T
T
Isobe
T
T
Ichimura
T
T
D048948:14-3-3 Proteins; D000818:Animals; D001933:Brain Stem / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D007700:Kinetics; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000302:isolation & purification / Q000378:metabolism*
D048948:14-3-3 Proteins; D009419:Nerve Tissue Proteins; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases