Phospholemman Is a Substrate for Myotonic Dystrophy Protein Kinase

Abstract

The genetic abnormality in myotonic muscular dystrophy, multiple CTG repeats lie upstream of a gene that encodes a novel protein kinase, myotonic dystrophy protein kinase (DMPK). Phospholemman (PLM), a major membrane substrate for phosphorylation by protein kinases A and C, induces Cl currents (I Cl(PLM) ) when expressed in Xenopus oocytes. To test the idea that PLM is a substrate for DMPK, we measured in vitro phosphorylation of purified PLM by DMPK. To assess the functional effects of PLM phosphorylation we compared I Cl(PLM) in Xenopus oocytes expressing PLM alone to currents in oocytes co-expressing DMPK, and examined the effect of DMPK on oocyte membrane PLM expression. We found that PLM is indeed a good substrate for DMPK in vitro. Co-expression of DMPK with PLM in oocytes resulted in a reduction in I Cl(PLM) . This was most likely a specific effect of phosphorylation of PLM by DMPK, as the effect was not present in oocytes expressing a phos(؊) PLM mutant in which all potential phosphorylation had been disabled by Ser 3 Ala substitution. The biophysical characteristics of I Cl(PLM) were not changed by DMPK or by the phos(؊) mutation. Co-expression of DMPK reduced the expression of PLM in oocyte membranes, suggesting a possible mechanism for the observed reduction in I Cl(PLM) amplitude. These data show that PLM is a substrate for phosphorylation by DMPK and provide functional evidence for modulation of PLM function by phosphorylation.Phosphorylation of membrane proteins by protein kinases is an important mechanism for receptor-mediated signal transduction in the regulation of cellular function. For instance, stimulation of ␤-adrenergic receptors activates protein kinase A (PKA) 1 , whereas stimulation of ␣1-adrenergic receptors activates protein kinase C (PKC). Though the end result of these kinases on cellular physiology can be very different, they share at least one membrane substrate, a 72-amino acid peptide with a single transmembrane domain called phospholemman (PLM) (1). Several lines of evidence suggest a role for PLM in ion transport and regulation of cell volume. In particular, expression of PLM in Xenopus oocytes leads to the appearance of a hyperpolarization-activated noninactivating Cl current (I Cl(PLM) ) (2-4). We have shown previously that the amplitude of I Cl(PLM) correlates with the level of PLM expression, and that co-expression of PKA increases both current amplitude and PLM expression (5). The highly conserved (6) cytoplasmic domain contains four potential phosphorylation sites, and loci for phosphorylation by PKA and PKC have been identified (1,7,8). PLM is also a substrate for NIMA ("never in mitosis") kinase, a product of the cell cycle regulatory gene nimA, which is necessary for cells to enter mitosis. PLM is, thus, a substrate for multiple kinases and may have a role as an integrator of adrenergic inputs.Myotonic muscular dystrophy, the commonest muscular dystrophy in adults, is an autosomal dominant-inherited multisystem disorder with prominent effects on skeletal and cardiac mu...

Effect of exercise and training on phospholemman phosphorylation in human skeletal muscle

“…Besides CaMKII, PKC could also play a role in PLM phosphorylation in response to exercise. PLM is known to be phosphorylated by PKC (33,42,51), and several studies have indicated that PKCs are activated during exercise (23,26,43,53). The PKC superfamily is divided into three subfamilies, conventional (␣-, ␤ I -, ␤ II -, and ␥-isoforms), novel (␦-, ε-, -, and -isoforms), and atypical PKCs (-and /-isoforms), based on differences in structure and responsiveness to the second messengers, Ca 2ϩ , and diacylglycerol (37).…”

“…Acute exercise in rat skeletal muscle induced phosphorylation and translocation of PLM to the muscle outer membrane fraction and sarcolemmal giant vesicles, with a concomitant increase in Na ϩ -K ϩ -ATPase activity (45). Insulin and exercise are hypothesized to promote PLM phosphorylation via protein kinase A (PKA) and C (PKC) since PLM is a known substrate for both PKA and PKC (33,42,51 (4,7). PKAand PKC-mediated phosphorylation of PLM may also regulate PLM translocation to the plasma membrane of the cell (27,34).…”

Cytoplasmic targeting signals mediate delivery of phospholemman to the plasma membrane

“…There was no change in the subcellular localization with either treatment (data not shown). Kinases other than PKA and PKC may act on PLM (29,37) and other FXYD family members. To determine whether phosphorylation of CHIF could affect its subcellular distribution, mutagenesis was performed on the carboxy terminus of CHIF (Table 2).…”

Phospholemman regulates cardiac Na+/Ca2+ exchanger by interacting with the exchanger's proximal linker domain

“…NMR (10) and infrared spectroscopy (2) showed that the TM domain of PLM reconstituted in liposomes is an ␣-helix with a maximum tilt of 15-17°. Specifically, NMR spectroscopic studies of highly purified PLM in model micelles indicate that the molecule consists of four ␣-helices: H1 (residues [12][13][14][15][16][17] is in the extracellular NH 2 terminus, H2 (residues [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] is the main TM helix followed by the short H3 (residues 39 -45), and H4 (residues 60 -68) in the COOH terminus is connected to H3 by a flexible linker (36). PKA phosphorylates Ser 68 , whereas PKC phosphorylates Ser 63 and Ser 68 , of PLM (37).…”

“…PKA phosphorylates Ser 68 , whereas PKC phosphorylates Ser 63 and Ser 68 , of PLM (37). In addition, PLM is also a substrate for myotonic dystrophy protein kinase (23) and never in mitosis A kinase (19). In transfected Madin-Darby canine kidney cells expressing mouse PLM, phosphorylation of Ser 69 is important in shifting the distribution of PLM from the endoplasmic reticulum to the plasma membrane (16).…”