PHOSPHORYLATION REACTIONS OF RECOMBINANT HUMAN MYOTONIC-DYSTROPHY PROTEIN-KINASE AND THEIR INHIBITION

The predicted protein kinase activity of the cloned gene product of th e human myotonic dystrophy locus has been experimentally verified. Aff inity-purified recombinant DM protein kinase became phosphorylated its elf and transphosphorylated histone H1. These activities were not pres ent in the bacterial host cells and were exhibited by DMPK and DMPKH, recombinant proteins which contain the protein kinase domain but exhib it distinct sizes, 43 and 66 kDa, respectively. DMPKH was further puri fied by velocity sedimentation on sucrose gradients; both activities m igrated with the recombinant protein at 41 S, consistent with discrete multimeric particles. Phosphoamino acid analysis showed that threonin e (predominantly) and serine were phosphorylated in both DMPKH and his tone H1. Although PKA and PKC are the known types of protein kinase wi th closest sequence homology to the DM protein kinase domain, purified DMPKH was inhibited by 4 mM but not 0.04-0.4 mM H7 and H8, which inhi bit PKA and PKC with K-i's of 0.4-15 mu M. Specific inhibitors of othe r classes of multifunctional serine/threonine protein kinases such as casein kinases I (CKI-7) and II (heparin) and calcium/calmodulin-depen dent protein kinase II (KN-62) did not inhibit DMPKH. DMPKH did not ph osphorylate membrane-associated phosphoproteins such as acetylcholine receptor or spectrin which are known to be substrates for PKA, PKC, an d CKI and -II, respectively. These experimental results suggest that t he active center of the recombinant human myotonic dystrophy protein k inase may have properties distinct from the well-studied classes of se rine/threonine protein kinases, in contrast to predictions based upon primary structure alone.