Calcium‐dependent protein kinase is localized with F‐actin in plant cells

Abstract

Abstract We recently purified a calcium‐dependent but calmodulin‐ and phospholipid‐independent protein kinase (CDPK) from cultured plant cells (Harmon et al.: Plant Physiology 83:830–837, 1987). A monoclonal antibody (mAb 3B9) directed against CDPK was used to localize this protein in Allium root cells and Tradescantia pollen tubes using immunofluorescence techniques. The mAb 3B9 staining pattern showed that CDPK is localized within a fibrous network in the cytoplasm resembling the normal interphase network of F‐actin. Treatment of tissue with 10 μM cytochalasin D (CD) prior to fixation abolished the staining pattern. Double‐localization experiments in which pollen tubes were first stained with mAb 3B9 and then with rhodamine‐phalloidin (RP) demonstrated that CDPK and F‐actin were colocalized. Monoclonal antibody 3B9 did not react with purified actin from rabbit muscle or Dictyostelium and did not bind to proteins corresponding to the M r of actin in crude extracts of Allium root tips and Tradescantia pollen tubes. CDPK did not phosphorylate purified rabbit muscle or Dictyostelium actin in vitro. Binding studies showed that CDPK (1) does not cosediment with actin filaments and (2) does not form a complex with G‐actin. The data indicate that although CDPK does not interact directly with actin, it may be associated with an actin‐binding protein and therefore could play a role in the regulation of the plant cytoskeleton.