Phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to increase the rate of phosphorylation of Ser(40).

Mark E Graham,Phillip W Dickson,Lia R.M Bevilaqua,Peter R Dunkley,Ellak I Von Nagy-Felsobuki

doi:10.1074/jbc.m105280200

Abstract

The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser(19) and Ser(40) produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser(19) phosphorylation is less pronounced than that of the Ser(40) phosphorylation. The effect of Ser(19) and Ser(40) phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser(19) and Ser(40) phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser(19) and Ser(40) were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser(40) is approximately 2- to 3-fold higher if Ser(19) is already phosphorylated. These results suggest that phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser(40) to kinases.

Highlights

Hydroxylase (TH) was studied directly using gel filtra- In vitro experiments have shown that a number of protein tion and indirectly using electrospray ionization mass kinases phosphorylate these residues [2]
Phosphorylation of proteins is one of the key elements involved in short term regulatory mechanisms in the cell
It could induce a subtle change in the structure of the protein to alter the substrate binding site but not change the overall shape of the molecule [30]

Summary

EXPERIMENTAL PROCEDURES

Materials—Vent DNA polymerase was from New England Biolabs. T4 DNA ligase was from Roche Molecular Biochemicals and the restriction enzymes from Promega. Radiochemicals, heparin-Sepharose, and calmodulin-Sepharose were from Amersham Pharmacia Biotech. The oligonucleotides were synthesized by Bresatec Ltd. The catalytic subunit of PKA was from Sigma. HPLC-grade acetonitrile and AnalaRgrade formic acid, methanol, and chloroform were from BDH Laboratein kinase; MOPS, 4-morpholinepropanesulfonic acid. This paper is available on line at http://www.jbc.org tory Supplies (Merck Pty. Limited, Kilsyth, Australia)

Tyrosine Hydroxylase Phosphorylation

RESULTS

TH ϩ Dopamine

DISCUSSION