Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play 
important roles in signal transduction required for cell proliferation, 
differentiation, and stress response and adaptation. It is also known that some 
prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in 
the characterization of these kinases is how to identify their specific 
substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 
from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly 
expressed in E. coli, cells are unable to form colonies on agar plates. This 
lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the 
highly conserved Lys residue was changed to Asn, indicating that phosphorylation 
of a cellular protein(s) in E. coli resulted in growth arrest. Several clones 
from an E. coli genomic library were found to suppress the lethal effect when 
co-expressed with pkn2. Four out of seven multi-copy suppressors were identified 
to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. 
Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, 
and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the 
phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part 
to the lethal effect in E. coli. The present method termed the STEK method 
(Suppressors of Toxic Effects of Kinases) may be widely used for the substrate 
identification not only for prokaryotic protein Ser/Thr kinases but also for 
eukaryotic kinases.