Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 pr oteins for optimal activation by phosphorylation. We examined the effects o f phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isofo rms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 ze ta and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 d id not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 in creased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding a ffinity to BMH1/BMH2 and sheep brain 14-3-3 increased. Both phosphoserine-s pecific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the ass ociation between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated T H decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of B MH1. These findings further support a role for 14-3-3 proteins in the regul ation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins.