The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide

Abstract

We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and paniculate (Triton X-100.solubilized) fractions and that upon gel filtration, both PTKs migrate as entiti~ of M, 44 009 [(1991) Biochem. Cell. Biol. 69. 146-153]. lterein we demonstrate by immunopre¢ipitation with anti-phosphotyrosine antibodies that the soluble PTK has the ability to undergo self-phosphory, lation. In addition, the polypeptide responsible for that ellzymatic activity has been identified by 2 approaches: (I) a two-dimensional elcetrophoresis, in which the first dimension performed in non-denaturing conditions allowed the localization of the native enzyme, while the second dimension ($DS-PAGE) permitted the analysis of alkali-resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS-PAGE followed by in situ phosphorylation (with [)'-'~"P]ATP) of polyGT el~trophoresed 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 he expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.

Recovery of Protein Kinases from Renatured SDS-Polyacrylamide Gels for Biochemical Studies

“…Phosphorylated peptides may also be subjected to phosphopeptide mapping. Although we did not detect tyrosine kinase activity in the present study using MBP as a substrate, tyrosine kinases were detected in studies using poly(Glu, Tyr) as a substrate in the in-gel kinase assay (6,7,10). It is reasonable to believe that tyrosine kinase activity is detectable by the procedure described here in a substrate-spe -cific manner.…”

“…Using different peptides, such as myelin basic protein (MBP), bacteria-produced c-Jun peptide and polydispersed co-polymer of glutamic acid and tyrosine [poly(Glu, Tyr)] as substrates immobilized in SDS-polyacrylamide gel (SDS-PAG), cellular proteins are electrophoresed. The kinase activity and molecular masses of many protein kinases were measured and detected, including MAPK/Erk (mitogen-activated protein kinase/extracellular signal regulated kinase) (9,16), SAMK/Krs1/2 (src-activated MBP kinase/kinases responsive to stress) (15,16), JNK/SAPK (Jun N-terminus kinase/stress activated protein kinase) (3) and several novel protein tyrosine kinases (6,7,10).…”

Capacitation Is Associated with Tyrosine Phosphorylation and Tyrosine Kinase-Like Activity of Pig Sperm Proteins1

“…Similar in-gel assays have been previously used to detect various kinases, e.g., microtubule-associated protein 2 kinase [57], histone kinase [58], and prostatic tyrosine kinase [59]. To our knowledge, the present research represents the first demonstration of a putative tyrosine kinase (or enzyme with tyrosine kinase-like activity) associated with capacitation in mammalian sperm.…”

Screening of Protein Tyrosine Kinases Activated during Neural Induction inXenopus

“…and was successfully used to identify PTKs in large Protein extracts (20 mg/lane) of uninduced and inamounts of tissue in dog and in human prostate (8,28). duced ectodermal explants from stage 9 and 11 emIn this study, we have shown that this method is very bryos were subjected to SDS-PAGE in the absence sensitive and can be applied to small samples such (Figs.…”