Antibodies to a Segment of Tyrosine Hydroxylase Phosphorylated at Serine 40 |
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Authors: | M. Goldstein,K. Y. Lee,J. Y. Lew,K. Harada,J. Wu,J. W. Haycock,&dagger T. Hokfelt, &Dagger A. Y. Deutch |
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Affiliation: | Neurochemistry Research Laboratory, New York University Medical Center, New York, New York, U.S.A.;; Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana, U.S.A.;; Department of Histology, Karolinska Institute, Stockholm, Sweden;and; Departments of Psychiatry and Pharmacology, Yale University Medical Center, New Haven, Connecticut, U.S.A. |
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Abstract: | Abstract: A synthetic peptide corresponding to residues 32–47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser40 and used to generate antibodies in rabbits. Reactivity of the anti-pTH32–47 antibodies with phospho- and dephospho-Ser40 forms of TH protein and peptide TH32–47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody-capture ELISAs, anti-pTH32–47 was more reactive with the phospho-TH than with the dephospho-TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho-TH forms. In western blots, labeling of the ∼60-kDa TH band by anti-pTH32–47 was readily detectable in lanes containing protein kinase A-phosphorylated native TH at 10–100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti-pTH32–47. Similarly, anti-pTH32–47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin enabled removal of TH activity by anti-pTH32–47. Lastly, in immunohistochemical studies, anti-pTH32–47 selectively labeled catecholaminergic cells in tissue sections from perfusion-fixed rat brain. |
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Keywords: | Tyrosine hydroxylase Protein phosphatase Antibody Phosphorylation of serine 40 Antiphosphorylated tyrosine hydroxylase Antidephosphorylated tyrosine hydroxylase |
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