Phosphorylation Reactions of Recombinant Human Myotonic Dystrophy Protein Kinase and Their Inhibition

Abstract

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.

Alternative Splicing Controls Myotonic Dystrophy Protein Kinase Structure, Enzymatic Activity, and Subcellular Localization

“…Reports on the (auto)phosphorylation activity of DMPK are scarce and mostly based on use of (improperly folded) bacterially expressed protein and/or standard, inefficient substrates like histone H1 or MBP (18,49,54). We demonstrate here that DMPK is a highly active kinase with a preferred substrate sequence consisting of a threonine (or serine) N-terminally preceded by three to four arginines (or lysines) at distinct positions.…”

Investigation of Myotonic Dystrophy Kinase Isoform Translocation and Membrane Association

“…Insect cells have been shown to faithfully localize and modify vertebrate proteins (25). Kinase activity has been demonstrated for DMK produced in bacteria (4). To ensure that DMK was also functional in insect cells, we performed a similar assay using histone H1 or myelin basic protein (MBP) as a substrate.…”

“…The removal of exons 12-15 greatly diminished the amount of the larger of the two complexes which migrated at the stacking gel interface. DMK expressed in bacteria sediments in sucrose gradients as large particles with a coefficient of 41 S, demonstrating that this aggregation was not specific to insect cells alone (4). The authors suggested a multimer of 21 subunits and observed 30 -50 nm spherical particles.…”

“…1 is an autosomal dominant heritable disorder caused by the expansion of a polymorphic (CTG) n repeat (1)(2)(3) found in the 3Ј-untranslated region of a serine/ threonine protein kinase (DMK) (4). It ranges in phenotype from mild adult-onset disorders to a severe congenital form that is frequently fatal after birth.…”

The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation.

“…The 15 residues that distinguish them from their closest relatives, the cyclic nucleotide-dependent kinases, are indicated in Figure 9. The human DM kinase (Dunne et al 1994) and the yeast DBF2/DBF20 kinases (Toyn and Johnston 1994) have been shown directly to have serine/threonine protein kinase activity, but their cyclic nucleotide dependence has not been reported. The similarities among the Wts-related kinases and the high degree of conservation of features critical to kinase function imply that these proteins may act in common pathways in vertebrates, invertebrates, and plants.…”