DIFFERENT SUSCEPTIBILITY OF PROTEIN-KINASES TO STAUROSPORINE INHIBITION - KINETIC-STUDIES AND MOLECULAR-BASES FOR THE RESISTANCE OF PROTEIN-KINASE CK2

A systematic analysis reveals that out of 20 protein kinases examined, specific for either Ser/Thr or Tyr, the majority are extremely sensit ive to staurosporine, with IC50 values in the low nanomolar range. A f ew of them however, notably protein kinases CK1 and CK2, mitogen-activ ated protein (MAP) kinase and protein-tyrosine kinase CSK, are relativ ely refractory to staurosporine inhibition, exhibiting IC50 values in the micromolar range. With all protein kinases tested, namely PKA, CK1 , CK2, MAP kinase (ERK-1), c-Fgr, Lyn, CSK and TPK-IIB/p38(Syk), staur osporine inhibition was competitive with respect to ATP, regardless of its inhibitory power. In contrast, either uncompetitive or noncompeti tive kinetics of inhibition with respect to the phosphoacceptor substr ate were exhibited by Ser/Thr and Tyr-specific protein kinases, respec tively, consistent with a different mechanism of catalysis by these tw o sub-families of kinases. Computer modeling based on PKA crystal stru cture in conjunction with sequence analysis suggest that the low sensi tivity to staurosporine of CK2 may be accounted for by the bulky natur e of three residues, Va166, Phe113 and Ile174 which are homologous to PKA Ala70, Met120 and Thr183, respec tively. In contrast these PKA res idues are either conserved or replaced by smaller ones in protein kina ses highly sensitive to staurosporine inhibition. On the other hand, H is160 which is homologous to PKA Glu170, appears to be responsible for the unique behaviour of CK2 with respect to a staurosporine derivativ e (CGP44171A) bearing a negatively charged benzoyl substituent: while CGP44171A is 10-100-fold less effective than staurosporine against PKA and most of the other protein kinases tested, it is actually more eff ective than staurosporine for CK2 inhibition, but it looses part of it s efficacy if it is tested on a CK2 mutant (H160D) in which His160 has been replaced by Asp. It can be concluded from these data that the ca talytic sites of protein kinases are divergent enough as to allow a co mpetitive inhibitor like staurosporine to be fairly selective, a featu re that can be enhanced by suitable modifications designed based on th e structure of the catalytic site of the kinase.