Phosphorylation of Jak2 by PKC-δ.

(A) IL-3–induced tyrosine phosphorylation of Jak2 in 32D-wt (left), 32D-δ (middle), and 32D cells expressing kinase-negative (KN) PKC-δ (right). Cells were treated with TPA and IL-3, as indicated. Jak2 was immunoprecipitated and subjected to anti-phosphotyrosine blotting. (B) Phosphorylation of Jak2 by PKC-δ. COS7 cells were transfected with HA-tagged Jak2 and PKC-δ and PKC-ε expression constructs as indicated, and Jak2 was immunoprecipitated with anti-HA antibody. The immunocomplexes were subjected to PKC kinase assay and resolved by SDS-PAGE before autoradiography. Expression of Jak2 was analyzed by anti-HA immunoblotting. (C) Cellular complex formation between PKC-δ and Jak2. HA-tagged Jak2 and PKC-δ were transfected to COS7 cells, as indicated. Cells were lysed in 1% NP40 lysis buffer. Jak2 was immunoprecipitated with anti-HA antibody, subjected to PKC kinase assay, and resolved by SDS-PAGE. Jak2, PKC-δ, and immunoglobulin-heavy chain are indicated by arrows. The band corresponding to PKC-δ–phosphorylated Jak2 protein was extracted from gel and subjected to phospho-amino acid analysis using 2-dimensional thin-layer electrophoresis.