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Fig. 1. Phosphorylation of golgin-160 by MLK3- and Rac-dependent pathways. (A) Phosphorylation of endogenous golgin-160 is enhanced in doxorubicin-treated cells. HeLa cells were metabolically labeled with ³⁵S-methionine and ³⁵S-cysteine for 2 hours and chased in normal growth medium in the absence or presence of doxorubicin (120 µM) for 4 hours. Immunoprecipitated golgin-160 was mock-treated or treated with protein phosphatase and visualized by fluorigraphy after SDS-PAGE. Elimination of the slower migrating forms in the presence of phosphatase indicates that golgin-160 is a phosphoprotein. Enhanced phosphorylation was observed in doxorubicin-treated cells. (B) Phosphorylation was examined by analysing slower-migrating forms of expressed golgin-160 in CHO cells by immunoblotting, using the anti-Myc (9E10) antibody, for Myc-tagged golgin-160 in cells transfected with wild-type MLK3 (WT) or catalytically inactive MLK3 (KR). The gel was run for 20 hours at 20 mA to enhance the separation of the phosphorylated forms of golgin-160. (C) Myc immunoblot of expressed golgin-160 in cells transfected with cDNA for wild-type (WT) or inactive (KR) MLK3 and inactive (N-) or active (V-) Rac. (D) Myc immunoblot of expressed golgin-160 in cells transfected with cDNA for v-Rac, dual leucine zipper-bearing kinase (DLK), wild-type MKK1 (WT) or a constitutively active MKK1 mutant. The upper and lower arrows in (B-D) indicate the position of the hyperphosphorylated (p) and hypophosphorylated (g160) forms of golgin-160, respectively. Data represent at least four experiments.

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