The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide.
We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and particulate (Triton X-100-solubilized) fractions and that upon gel filtration, both PTKs migrate as entities of Mr 44,000 [(1991) Biochem. Cell. Biol. 69, 146-153]. Herein we demonstrate by immunoprecipitation with anti-phosphotyrosine antibodies that the soluble PTK has the ability to undergo self-phosphorylation. In addition, the polypeptide responsible for that enzymatic activity has been identified by 2 approaches: (1) a two-dimensional electrophoresis, in which the first dimension performed in non-denaturing conditions allowed the localization of the native enzyme, while the second dimension (SDS-PAGE) permitted the analysis of alkali-resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS-PAGE followed by in situ phosphorylation (with [gamma-32P]ATP) of polyGT electrophoresed together with the enzyme preparation; the exclusive presence of the radiolabeled phosphotyrosine in the renatured protein confirmed its enzymatic nature. Using these methods, the major form of PTK in the dog prostate was shown to be expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.
Department of Biochemistry, University of Montreal, Canada.
L T
LT
Nguyen
Y
Y
Durocher
A
A
Chapdelaine
S
S
Chevalier
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional; D007526:Isoelectric Point; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D019000:Phosphotyrosine; D011467:Prostate / Q000201:enzymology*; D011489:Protein Denaturation; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology
D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases