Figure 3.

Specific interaction between ZIP and PKC-ζ. (A) GST control protein or a GST-ZIP fusion protein (as indicated) was immobilized on glutathione-Sepharose beads and incubated with purified PKC-ζ (lane 2), with purified PKC-α (lane 3) or purified PKC-βI (lane 4). After washing the complexes were resolved by SDS/PAGE and the same immunoblot was sequentially incubated with antiserum against PKC-ζ (top) or PKC-α and -β (bottom). Lanes 5–7 show the amounts of purified PKC proteins used in the binding assays. (B) PKC-ζ purified from insect cells was incubated in a phosphorylation reaction with [³²P]-labeled ATP either alone (lane 1), with purified GST-ZIP (lane 3) or with an unrelated control protein GST-MEK (lane 2). Shown is an autoradiograph of the phosphorylation reaction. (C) PKC-ζ was immunoprecipitated from extracts of COS cells expressing ZIP (lane 1), PKC-ζ (lane 2), or both ZIP and PKC-ζ (lane 3). The immunoprecipitates were analyzed for the presence of PKC-ζ (Left) or ZIP (Right) by sequential Western blot analysis.