Two Ser/Thr protein kinases, SpkA and SpkB, selected from Myxococcus xanthus based on amino acid sequence similarities with the catalytic subunits of cAMP-dependent protein kinases (PKA) were synthesized using a cell-free protein synthesis system. in various protein kinase assays, purified StkA and StkB showed their highest protein kinase activities in a PKA assay using the selective PKA substrate Kemptide and in a protein kinase C (PKC) assay using the selective PKC substrate neurogranin((28-43)), respectively. SpkA had apparent K-m values of 45 mu M and 37 mu M for Kemptide and ATP, respectively. Phosphorylation of Kemptide was inhibited by a specific PKA inhibitor peptide, PKI5-24, and the IC50 and K-i values for inhibition of the SpkA activity were 117 nM and 36 nM, respectively. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.