Phosphorylation reactions of recombinant human myotonic dystrophy protein kinase and their inhibition.
The predicted protein kinase activity of the cloned gene product of the human myotonic dystrophy locus has been experimentally verified. Affinity-purified recombinant DM protein kinase became phosphorylated itself and transphosphorylated histone H1. These activities were not present in the bacterial host cells and were exhibited by DMPK and DMPKH, recombinant proteins which contain the protein kinase domain but exhibit distinct sizes, 43 and 66 kDa, respectively. DMPKH was further purified by velocity sedimentation on sucrose gradients; both activities migrated with the recombinant protein at 41 S, consistent with discrete multimeric particles. Phosphoamino acid analysis showed that threonine (predominantly) and serine were phosphorylated in both DMPKH and histone H1. Although PKA and PKC are the known types of protein kinase with 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 inhibit PKA and PKC with Ki's of 0.4-15 microM. Specific inhibitors of other classes of multifunctional serine/threonine protein kinases such as casein kinases I (CKI-7) and II (heparin) and calcium/calmodulin-dependent protein kinase II (KN-62) did not inhibit DMPKH. DMPKH did not phosphorylate membrane-associated phosphoproteins such as acetylcholine receptor or spectrin which are known to be substrates for PKA, PKC, and CKI and -II, respectively. These experimental results suggest that the active center of the recombinant human myotonic dystrophy protein kinase may have properties distinct from the well-studied classes of serine/threonine protein kinases, in contrast to predictions based upon primary structure alone.
Dunne
P W
PW
Department of Neurology, Baylor College of Medicine, Houston, Texas 77030.
Walch
E T
ET
Epstein
H F
HF
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D065636:Myotonin-Protein Kinase; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship
C585517:DMPK protein, human; D017931:DNA Primers; D011994:Recombinant Proteins; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases