LYN kinase expression and activity in K562 and K562-R cells.

(A) K562-R cell lysates (250 μg) were immunodepleted with anti-LYN (2 μg) or anti-HCK (2 μg) and protein A/G–sepharose, and resultant supernatants were analyzed for total tyrosine phosphoproteins (p-Tyr; left) or LYN (right). Migration of protein standards is shown on the left, and the 2 forms of LYN expressed in K562-R cells are shown on the right. (B) Twenty micrograms RNA from K562 and K562-R cells was resolved by electrophoresis, transferred to a membrane, and hybridized with a LYN probe as described in “Materials and methods.” The extent of hybridization with probe is shown (top), and the migration of 28S and 18S RNA is shown at the bottom. (C) Equal protein (30 μg) K562 and K562-R cell lysates were subjected to immunoblotting for HCK (left) and LYN (right). U-937 cell lysates were used as a positive control for HCK. Two forms of LYN (but not HCK) were detected in K562 cells, and the p53 form of LYN was overexpressed in K562-R cells. The same blots were probed with antiactin to monitor protein loading. (D) LYN or HCK immune complexes from K562 and K562-R cells were analyzed for tyrosine kinase activity in the presence of [³²P]-ATP and enolase as described in “Materials and methods.” Src immune complexes from HT-29 cells were used as a positive control. Phosphorylation was detected by autoradiography on x-ray film. LYN autophosphorylation was detected in K562-R immune complexes, while exogenous substrate (enolase) phosphorylation was detected in both K562 and K562-R immune complexes. K562-R cell enolase phosphorylation was estimated (by PhosphorImager) to be approximately 7-fold higher than K562 cells.