By combination of column chromatographies (heparin-agarose, HiTrap heparin and HiTrap SP columns) and gel filtration on a Superdex 200-pg HPLC column, an actin kinase was partially purified from a 1.5 M NaCl extract of porcine liver. The actin kinase was finally purified, by actin-Sepharose column chromatography (HPLC), as an actin-binding protein kinase. The biochemical properties, such as (1) requirements of divalent cations (10 mM Mg2+ and 3 mM Mn2+) and effective phosphate acceptors (actin and α-casein), (2) phosphorylation of both Ser- and Thr-residues on these two phosphate acceptors, (3) autophosphorylation of the catalytic subunit (approximately 37 kDa), and (4) inhibition kinetics by CK-I-7 (a CK-I specific inhibitor), of the purified actin kinase were similar to those reported for CK-I purified from various mammalian cells, but it was distinguishable from three cellular actin kinases (A-kinase, C-kinase and actin-fragmin kinase (approximately 80 kDa)). The 37 kDa actin kinase-mediated phosphorylation of actin did not relate to its polymerizability. Inhibition of CK-II-mediated phosphorylation of functional cellular proteins, including calmodulin (CaM), by actin was significantly stimulated after its full phosphorylation by the purified 37 kDa actin kinase or rCK-I in vitro. These results suggest that: (1) the 37 kDa Ser/Thr actin-binding kinase may be classified as a member of the CK-I family; and (2) specific phosphorylation of actin by the actin kinase may be involved in the suppression mechanism of CK-II-mediated signal transduction at the cellular level. Copyright (C) 1999 Elsevier Science B.V.
All Science Journal Classification (ASJC) codes
- Molecular Biology