Phosphorylation and activation of tryptophan hydroxylase 2: identification of serine-19 as the substrate site for calcium, calmodulin-dependent protein kinase II.
Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the biosynthesis of serotonin. TPH was once thought to be a single-gene product but it is now known to exist in two isoforms. TPH1 is found in the periphery and pineal gland whereas TPH2 is expressed specifically in the CNS. Both TPH isoforms are known to be regulated by protein kinase-dependent phosphorylation and the sites of modification of TPH1 by protein kinase A have been identified. While TPH2 is activated by calcium, calmodulin-dependent protein kinase II (CaMKII), the sites at which this isoform is modified are not known. Treatment of wild-type TPH2 with CaMKII followed by mass spectrometry analysis revealed that the enzyme was activated and phosphorylated at a single site, serine-19. Mutagenesis of serine-19 to alanine did not alter the catalytic function of TPH2 but this mutant enzyme was neither activated nor phosphorylated by CaMKII. A phosphopeptide bracketing phosphoserine-19 in TPH2 was used as an antigen to generate polyclonal antibodies against phosphoserine-19. The antibodies are highly specific for phosphoserine-19 in TPH2. The antibodies do not react with wild-type TPH2 or TPH1 and they do not recognize phophoserine-58 or phosphoserine-260 in TPH1. These results establish that activation of TPH2 by CaMKII is mediated by phosphorylation of serine-19 within the regulatory domain of the enzyme. Production of a specific antibody against the CaMKII phosphorylation site in TPH2 represents a valuable tool to advance the study of the mechanisms regulating the function of this important enzyme.
Kuhn
Donald M
DM
Department of Psychiatry & Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, Michigan, USA. donald.kuhn@wayne.edu
Sakowski
Stacey A
SA
Geddes
Timothy J
TJ
Wilkerson
Curtis
C
Haycock
John W
JW
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020134:Catalytic Domain / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D014365:Tryptophan Hydroxylase / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D012694:Serine; C487802:Tph2 protein, mouse; D014365:Tryptophan Hydroxylase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2