Phosphorylation of human recombinant tyrosine hydroxylase isoforms 1 and 2: an additional phosphorylated residue in isoform 2, generated through alternative splicing.
The single human tyrosine hydroxylase (TH) gene generates four different mRNA species through alternative splicing events. TH-1 and TH-2 mRNAs are expressed mostly in the brain. We have produced large amounts of the corresponding proteins in Escherichia coli to analyze their respective molecular characteristics. The polypeptides have molecular weights similar to those of TH expressed in Xenopus oocytes and react with antibodies to TH. The two isoforms were purified with a purity of 90% using a three-step procedure. The phosphorylation sites have been determined in the two isoforms after labeling with [gamma-32P]ATP in the presence of cAMP-dependent protein kinase (PKA) or calmodulin-dependent protein kinase II (CaM-PK II). In both isoforms, Ser-40 was found to be phosphorylated by PKA, and Ser-19 and Ser-40 were found to be phosphorylated by CaM-PK II. The putative phosphorylation site generated by alternative splicing (Ser-31) was phosphorylated specifically by CaM-PK II in TH-2 only. The kinetic properties of the two isoforms in the presence of various concentrations of the substrate (tyrosine) and of the natural cofactor [6R)-tetrahydrobiopterin) were also analyzed. TH produced in E. coli is unphosphorylated but nevertheless active. At 50 microM tyrosine and 300 microM (6R)-tetrahydrobiopterin, the specific activities of TH-1 and TH-2 are 1300 and 620 nmol of dihydroxyphenylalanine/min/mg, respectively. Phosphorylation of TH-1 and TH-2 by PKA activates both isoenzymes as shown by the increase in the affinity for the cofactor. No changes in kinetic parameters of the isoenzymes were observed after phosphorylation by CaM-PK II. Dopamine was found to inhibit both TH isoenzymes to the same extent as shown by their similar Ki values for dopamine. These values were increased after phosphorylation of each enzyme by PKA. Unlike TH-1, phosphorylation of TH-2 by CaM-PK II resulted in an increase of the Ki value for dopamine. This property may be related to the presence of the additional phosphorylated residue in TH-2 isoform.
Laboratoire de Neurobiologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
B
B
Le Bourdellès
P
P
Horellou
J P
JP
Le Caer
P
P
Denèfle
M
M
Latta
J
J
Haavik
B
B
Guibert
J F
JF
Mayaux
J
J
Mallet
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003854:Deoxyribonucleotides; D004298:Dopamine / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D003854:Deoxyribonucleotides; D007527:Isoenzymes; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D004298:Dopamine