Antibodies to a segment of tyrosine hydroxylase phosphorylated at serine 40.
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 approximately 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.
Goldstein
M
M
Neurochemistry Research Laboratories, New York University Medical Center, NY 10016, USA.
Lee
K Y
KY
Lew
J Y
JY
Harada
K
K
Wu
J
J
Haycock
J W
JW
Hokfelt
T
T
Deutch
A Y
AY
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D005576:Colforsin / Q000494:pharmacology; D003342:Corpus Striatum / Q000196:embryology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D008297:Male; D016716:PC12 Cells / Q000201:enzymology; D010766:Phosphorylation; D010768:Phosphoserine / Q000276:immunology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000276:immunology; D013574:Synaptosomes / Q000201:enzymology; D014446:Tyrosine 3-Monooxygenase / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D011994:Recombinant Proteins; D010768:Phosphoserine; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D014446:Tyrosine 3-Monooxygenase; D017868:Cyclic AMP-Dependent Protein Kinases