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J. Biol. Chem., Vol. 275, Issue 30, 23362-23367, July 28, 2000

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Phospholemman Is a Substrate for Myotonic Dystrophy Protein Kinase^*

J. Paul Mounsey^§, J. Edward John III, Steve M. Helmke^¶, Erik W. Bush^¶, John Gilbert, Allen D. Roses, M. Benjamin Perryman^¶, Larry R. Jones^**, and J. Randall Moorman

From the  Departments of Internal Medicine (Cardiovascular Division), Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, ^¶ Department of Medicine, Division of Cardiology, University of Colorado Health Science Center, Denver, Colorado 80262,  Departments of Internal Medicine (Neurology) and Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, and the ^** Departments of Internal Medicine and Pharmacology, Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana 46202

Received for publication, February 3, 2000, and in revised form, April 11, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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  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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 muscle. The genetic abnormality is amplification of a CTG repeat sequence in the 3'-untranslated region of the dmpk gene in chromosome 19q13.3, which encodes a novel protein kinase, DMPK (9-13). Substrates for DMPK include myogenin and the -subunit of the L-type calcium channels (14, 15). Little is known, however, of other substrates, especially those involving transmembrane ion fluxes and membrane excitability.

We have tested the hypothesis that PLM is a substrate for DMPK because 1) PLM is a major substrate for other protein kinases and 2) PLM forms or regulates Cl channels in Xenopus oocytes. To test our hypothesis, we first assessed whether PLM was a substrate for DMPK in vitro. We subsequently measured the amplitudes and biophysical characteristics of Xenopus oocyte-expressed I_Cl(PLM) in the presence and absence of the mRNA-encoding DMPK. Finally we measured the effect of DMPK co-expression on the level of PLM expression in oocyte membranes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DMPK Phosphorylation Assays

cDNA Constructs and Fusion Protein Expression-- A full-length 1890-base pair coding sequence, only DMPK cDNA derived from the complete and correct DMPK cDNA (GenBank^TM accession number L08835) (16) was cloned in-frame into the EcoRI site of the prokaryotic expression vector pGEX-6P-1 (Amersham Pharmacia Biotech), which appends a GST affinity tag to the amino terminus of DMPK. GST-DMPK subclones were rarely recovered in the correct orientation, which is a previously reported observation (17). Full-length recombinant GST-DMPK protein was poorly expressed in a variety of Escherichia coli strains (data not shown), possibly because of toxicity caused by the hydrophobic transmembrane domain. A carboxyl-terminal DMPK deletion clone (GST-tDMPK) lacking amino acids 545-629, including the transmembrane domain, was derived from the full-length construct and could be expressed in TOP10F' cells (Invitrogen).

Purification of GST-tDMPK-- Cultures of E. coli transformed with GST-tDMPK were induced with 1 mM isopropyl-1-thio--D-galactopyranoside (Sigma), grown at 30^o for 4 h, and harvested by pelleting. Cell pellets were resuspended in 50 mM Tris, pH 8.0, 0.2 M NaCl, 5 mM dithiothreitol, 2 mM EDTA, 1 mM AEBSF, 0.1 mM leupeptin, and 10 µg/ml aprotinin (all from Sigma) and lysed by the addition of 1 mg/ml lysozyme (Sigma) for 20 min on ice followed by the addition of 1% TX100 (Pierce, Surfact-Amps X-100) for 10 min on ice. All subsequent steps were performed at 4 °C. The lysate was centrifuged at 18,000 × g for 30 min, and the supernatant was incubated overnight with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). The beads were washed four times with cold 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM dithiothreitol, and 2 mM EDTA. GST-tDMPK was eluted from the beads by the addition of 50 mM Tris, pH 8.0, 10 mM glutathione (Sigma), 5 mM dithiothreitol, 2 mM EDTA, 1 mM AEBSF, 0.1 mM leupeptin, and 10 µg/ml aprotinin, analyzed on Coomassie Blue-stained SDS gels, and used directly in kinase assays.

Kinase Assays-- Kinase assays were performed in a volume of 40 µl with 10 mM MgCl₂ (Sigma), 0.1 mM [-³²P]ATP (1.5 mCi/µmole) (NEN Life Science Products) and the indicated amount of enzyme and substrate for 1 h at 37 °C. Purified phospholemman was used at 10 µM final concentration (3.4 µg in 40 µl). The PKC substrates used were the glycogen synthase residue 1-8 peptide analog, PLSRTLSVAAKK, referred to as the glycogen synthase peptide, and the epidermal growth factor receptor residues 650-658 peptide, VRKRTLRRL, referred to as the epidermal growth factor receptor peptide (18) and myelin basic protein. Reactions were started with the addition of Mg[-³²P]ATP and stopped with a modified Tris-Tricine gel sample buffer. Samples were heated to 100 °C for 5 min, cooled, loaded onto precast 10-20% acrylamide Tris-Tricine gels (Bio-Rad), and electrophoresed for 3 h at 75 V. Resolution of substrates on Tris-Tricine gels permits the unambiguous determination of protein phosphorylation as opposed to filter binding assays in which spurious counts can be introduced from autophosphorylated kinases and contaminants. Gels were treated for 30 min with 10% glutaraldehyde (Fisher, reagent grade) to fix the proteins and extensively washed to remove [-³²P]ATP. Phosphorylated proteins were visualized with a Molecular Dynamics PhosphorImager, and the loading equivalency was verified by Coomassie Blue staining. Coomassie Blue-stained phosphoproteins were excised from the gels and quantitated by liquid scintillation counting. Specific phosphorylation activities were calculated after subtracting the background counts found in the substrates incubated in the absence of GST-tDMPK from the total counts found in the presence of GST-tDMPK.

Molecular Reagents and Immunoblotting

Wild-type and mutant PLM and DMPK mRNAs were prepared as described previously (19, 20), and all new mutant PLM cDNAs were dideoxy-sequenced. Immunoblotting and preparation of antibodies were performed as described previously (2, 20, 21).

Oocyte Expression and Electrophysiology

Methods for oocyte handling and injection have been described (2, 4, 5, 19, 22). Briefly, stages IV and V Xenopus oocytes were isolated manually in modified Barth's solution containing penicillin and streptomycin, immediately after excision from the frog. The oocytes were not exposed to collagenase. Injection of RNA (4-8 ng of wild type or phos() PLM, and 50 ng of wild type DMPK mRNA, in 100 mM KCl) was accomplished using a graduated, mechanical 10-ml micropipettor (Drummond, Broomall, PA). Within each batch of oocytes, the amounts of wild type and phos() mRNA were the same. Injected oocytes were incubated in plastic Petri dishes and continuously rotated in an incubator at 19 °C. The oocytes were defolliculated manually the day of the electrophysiologic experiments, 2-5 days after injection.

Whole cell recordings were obtained 1-8 days after injection using a two-microelectrode voltage clamp (oocyte clamp OC-725, Warner Instruments, New Haven, CT) at room temperature as described previously (4, 19). Current and voltage electrodes were filled with 3 M KCl and 10 mM HEPES (pH 7.35). The bath solution contained 147 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 10 mM glucose, 10 mM HEPES, pH 7.4 (NaOH); the flow rate was 3-4 ml/min, and the bath chamber volume was 2 ml. Voltage protocols were delivered, and currents were digitized using pCLAMP (Axon Instruments) hardware and software. Oocytes were held at 10 mV. Two-second hyperpolarizing clamp pulses from 40 to 170 mV were applied in 10 mV increments; tail currents were recorded at 40 mV. Current amplitudes were measured as the difference in current levels from the beginning to the end of the hyperpolarizing pulses and were normalized for each batch of oocytes by dividing by the mean current amplitude in oocytes expressing wild type (or phos()) PLM alone at a test potential of 170 mV (for the current-voltage relationships) or 150 mV (for the box plots of current amplitudes). Tail current amplitudes were normalized by subtracting the linear component of leak current and then dividing by the tail current amplitude after a hyperpolarizing step to 170 mV. Data were fit using a nonlinear least squares method (NFIT, Island).

Data Analysis

Statistical tests were performed with SigmaStat (Jandel). The data in Fig. 3 did not pass a Kolmogorov-Smirnov test for normality, and nonparametric statistical tests were employed. Plots were prepared with Origin (Microcal) and SigmaPlot (Jandel).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PLM Is a Substrate for DMPK-- GST-tDMPK phosphorylated PLM (Fig. 1). The specific activity for the phosphorylation of PLM was high at 78 pmol/min/mg. Other substrates phosphorylated by GST-tDMPK included phospholamban, the glycogen synthase peptide, the epidermal growth factor receptor peptide, and myelin basic protein (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   GST-tDMPK phosphorylates phospholemman. Kinase assays were performed using 50 ng of GST-tDMPK and 3.4 µg of PLM. Reactions were stopped with a modified sample buffer, and the samples were resolved on 10-20% acrylamide Tris-Tricine gels and fixed with glutaraldehyde. Phosphorylation was visualized by PhosphorImager and loading equivalency verified by Coomassie Blue staining (not shown).

Co-injection of DMPK mRNA Reduces I_Cl(PLM)-- Fig. 2 shows examples of families of ion currents elicited by stepwise hyperpolarization of oocytes isolated from the same frog. Each record shows the effect of hyperpolarization from a holding potential of 10 mV to between 30 and 170 mV for 2 s followed by repolarization to 40 mV. Expression of PLM alone (Fig. 2A) induced large hyperpolarization-activated currents, which we have previously shown to be carried by Cl and called I_Cl(PLM) (23). The important finding was that co-injection of DMPK mRNA led consistently to a reduction in the amplitude of I_Cl(PLM) (Fig. 2B). In these oocytes, the endogenous hyperpolarization-activated Cl current, which we have called I_Cl(endo) (4), was small (Fig. 2C), and the injection of DMPK mRNA alone did not affect I_Cl(endo) amplitude (not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   AE, families of ICl(PLM) current amplitudes were measured as the difference in current levels at the beginning and end of the traces at a test potential of 150 mV, as shown by the arrows in B. Holding potential was 10 mV (near the resting potential). F, time course of ICl(PLM) activation. The Fig. shows superimposed averaged, normalized currents from oocytes expressing PLM alone (solid line) and oocytes co-expressing DMPK (dotted line) The coefficient of variation at 50% maximal current was less than 10%.

The results of a series of these experiments are shown in the first two box plots of Fig. 3. To facilitate comparison of oocytes isolated from different frogs, with different levels of PLM expression, currents have been normalized so that oocytes expressing PLM alone have a current of 1.0. In 122 oocytes (15 batches) co-expressing DMPK, the mean normalized currents were reduced to 0.54 compared with 136 oocytes expressing PLM alone (p < 0.0001, Mann-Whitney rank sum test).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Box plots of ICl(PLM) amplitudes. In the box plot symbol, the dotted horizontal line is the mean; the solid horizontal line is the median; the box encloses 50% of the data, and the hatches enclose 80%. The number of oocytes (frogs) in the boxes from left to right are 136 (16), 122 (15), 120 (15), and 103 (12).

The DMPK Effect Was Not Present in a Phosphorylation-deficient PLM-- There are four potential phosphorylation sites in PLM, Ser⁶², Ser⁶³, and Ser⁶⁸, and Thr⁶⁹. To determine whether the effect of DMPK mRNA expression required phosphorylation of PLM, we measured the amplitude of Cl currents induced by expression of a mutated PLM molecule in which these phosphorylation sites had been disabled by Ser or Thr mutation to Ala (S62A/S63A/S68A/T69A; phos (-)). We reasoned that any nonspecific effect of DMPK to reduce oocyte translation of heterologous mRNAs would be preserved in these oocytes but that PLM-specific phosphorylation would be prevented. As shown in Fig. 2D, the mutated phos() PLM mRNA induced I_Cl(PLM) of amplitude equivalent to wild type after the injection of the same amount of mRNA and the same incubation period. The important finding was that the effect of co-injecting DMPK mRNA was lost on the phos() mutated PLM (Fig. 2E). This demonstrates that phosphorylation of PLM is necessary for DMPK to exert an effect on I_Cl(PLM).

As shown in Fig. 3, the results of experiments in 12 batches of oocytes showed the mean normalized phos() I_Cl(PLM) was not significantly changed by co-expression of DMPK (n = 120 and 103; p = not significant, Mann-Whitney rank sum test). The amplitude of I_Cl(PLM) in oocytes injected with mutated phos() mRNA did not differ significantly from control.

Phosphorylation of PLM Does Not Affect Biophysical Characteristics of I_Cl(PLM)-- One mechanism by which phosphorylation might have reduced currents at a given test potential is by altering the voltage sensitivity of channel opening. This has been shown to be the case for neuronal Ca²⁺ currents in the presence of adrenergic agents, where currents elicited by large depolarizations were less reduced than those at smaller depolarizations (24). The current-voltage relationship in Fig. 4A argues that DMPK does not have such an effect on PLM channels over the voltage range measured.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Characteristics of ICl(PLM) activation. A and B, normalized current-voltage relationships for wild type (A) and phos() ICl(PLM). Error bars are S.E. and are omitted if smaller than the symbols. C, voltage sensitivity of activation gating. The peak amplitudes of tail currents at 40 mV are plotted as a function of the potential of a 2-s conditioning pulse from a holding potential of 10 mV. The tail current amplitudes were normalized to the largest one for each oocyte, and the averages of eight to ten are shown. Error bars are S.E. and are omitted if smaller than the symbols. The slopes through the points representing 1-10% of maximum current are essentially identical.

Interestingly, the current-voltage relationship of currents induced by expression of phos() PLM did not differ from those induced by wild type PLM. This suggests that any PLM sites phosphorylated by the oocyte play no detectable role in the voltage dependence of I_Cl(PLM). To resolve the voltage sensitivity of the currents further, we estimated the gating valence (4, 25). Fig. 4C is a plot of the log of the normalized tail current amplitude at a potential of 40 mV as a function of the conditioning 2-s hyperpolarizing step. The ordinate is interpreted as the probability of channel opening (P_o) during the hyperpolarizing step. The gating valence z, the minimum number of charges traversing the full thickness of the membrane during channel opening, is related to the slope of the line, log P_o/V. The straight line is of the form,

(Eq. 1)

where R, T, and F have their usual meanings. The estimated gating valences are also given in Table I and do not differ significantly from each other.

Another mechanism by which phosphorylation might reduce current amplitude is by slowing activation kinetics. Fig. 2F shows examples of averaged, normalized currents from oocytes expressing PLM alone and from oocytes co-expressing DMPK at a test potential of 150 mV. Each is derived from 10 to 15 traces from two or more frogs. The time courses of activation appear much the same, suggesting that phosphorylation did not affect activation kinetics significantly. These traces, and others from phos() PLM were fit to the expression,

(Eq. 2)

where I is current at time t, and A, B, N, C, and R are constants. The coefficients are given in Table I, as are the times to half-maximal activation, a model-independent measure of activation. None are significantly different from one another. Thus we find no effect on the voltage dependence of wild type PLM Cl current activation by co-expression of DMPK.

Phosphorylation of PLM by DMPK Reduced PLM Expression in Xenopus Oocyte Membranes-- Fig. 5 shows an immunoblot probed with affinity purified polyclonal antibodies to PLM 58-72, the 15-carboxyl-terminal amino acids of PLM. Each lane contains membranes from five oocytes previously used for measurement of current amplitude. The first lane shows oocytes expressing wild-type PLM alone and the second lane shows oocytes co-expressing DMPK. Consistent with the reduction in current amplitude the amount of immunoreactive PLM was greater in the oocytes expressing PLM alone compared with the oocytes co-expressing DMPK. The result was confirmed in one other immunoblot and was the same using affinity purified antibodies to PLM 1-15, the 15-amino-terminal amino acids. These data indicate that the reduction of I_Cl(PLM) was accompanied by a decrease in PLM expression in the oocyte membrane.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Immunoblot of oocyte membranes probed with polyclonal anti-PLM antibodies. Left lane, membranes from oocytes injected with PLM mRNA alone; right lane, oocytes co-injected with DMPK mRNA. Note the reduction in PLM expression in the oocytes co-expressing DMPK. Membranes from five oocytes previously used for current measurements were studied for each condition.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To assess whether PLM is a substrate for DMPK, we have measured phosphorylation of PLM by DMPK in vitro. To assess the potential functional effects of PLM phosphorylation, we measured Cl currents induced by PLM expression in Xenopus oocytes and oocyte membrane PLM expression. Our most important findings are 1) PLM is a good substrate for DMPK in vitro; 2) co-expression of the normal DMPK with wild type PLM reduces I_Cl(PLM) by about half; 3) this effect is because of PLM phosphorylation, because the effect is lost if the phosphorylation sites of PLM are disabled by site-directed mutations; and 4) the reduction in current amplitude results from a reduction in oocyte membrane PLM expression.

The functional effect of DMPK co-expression in Xenopus oocytes was a reduction in I_Cl(PLM). This reduction was abolished in a PLM mutant in which all the potential phosphorylation sites in the cytoplasmic domain of the molecule had been disabled by site-directed mutagenesis. This observation, taken with the results of in vitro phosphorylation experiments, suggests strongly that the effects of DMPK on I_Cl(PLM) were the result of PLM phosphorylation. Co-expression of PKC also causes a reduction of I_Cl(PLM), but dissimilar to the effects of DMPK, disabling the PLM phosphorylation sites had no effect on the response to PKC (5). PKA co-expression causes an increase in I_Cl(PLM) sites, which is dependent on the presence of intact PLM phosphorylation (5). This increase in I_Cl(PLM) is accompanied by an increase in PLM expression in the Xenopus oocyte membrane. Similar to the mechanism of PKA, the mechanism of the reduction in I_Cl(PLM) induced by DMPK co-expression appeared to be a reduction in PLM expression. The biophysical properties of the current were unaffected by DMPK.

Patients with myotonic muscular dystrophy have abnormalities of Na²⁺ currents (26) that might lead to myotonia (27), and mice with DMPK deficiency have similarly abnormal Na²⁺ channel gating.² A defect of Na²⁺ conductance may not be the only membrane electrophysiological abnormality in skeletal muscle in DM, however. Myotonia can be induced in normal skeletal muscle preparations by maneuvers that would reduce membrane Cl conductance, e.g. Cl transport blockers (29) or removal of external Cl (30). Patients with dominant and recessive human myotonia and myotonic goats have reduced sarcolemmal Cl conductance (31-34). Dominant and recessive human myotonia (35) and mouse myotonia (36) are linked to abnormalities of a Cl channel gene. Because Cl is the most permeant anion in skeletal muscle membranes, Cl conductance serves to oppose excitability; reduced Cl conductance results in myotonia. In patients with myotonic dystrophy, Cl conductance may be either normal or reduced, and membrane potential may be normal or depolarized (37, 38). The heterogeneity of the clinical syndrome may relate in part to this variability. Franke et al. (26) noted that the patients with myotonia had reduced Cl conductance and normal membrane potentials, whereas patients with predominantly dystrophic manifestations had normal Cl conductance and depolarized membrane potentials.

Cl channels play important roles not only in regulation of excitability but also in regulation of cell water transport and cell division. Homologues of PLM also have roles in these physiological processes. The -subunit of the Na,K-ATPase co-precipitates with the a and b subunits and modulates pump activity (39). A role in transmembrane ion and water movement has been suggested, as it is required for murine blastocyst cavitation (40), and it is localized to kidney tubule membrane (39). Mammary-associated tumor 8 is expressed in mouse breast cancers initiated by the transgenic oncogenes (41). When expressed in Xenopus oocytes, mammary-associated tumor 8 induces hyperpolarization-activated currents similar to those induced by PLM (42). Related ion channel is one of 12 genes induced by the oncoprotein E2a-PbxI in NIH3T3 fibroblasts (43). We have found that related ion channel expression induces hyperpolarization-activated currents in RNA-injected Xenopus oocytes.³ Channel-inducing factor was isolated from colons of rats after mineralocorticoid treatment (44) and is constitutively expressed in kidney, where it is up-regulated by aldosterone (45). Interestingly, its expression in Xenopus oocytes leads to depolarization-activated K²⁺ currents (44). Whereas the function of each family member may differ, they appear to share a common mechanism of action in regulation of transmembrane ion, osmolyte, and water flux.

Interestingly, DMPK belongs to a new family of kinases, including the DBF2 (46) and DBF20 (47) kinases in Saccharomyces cerevisiae, Warts kinase in Drosophila melanogaster (48), Cot-1 kinase in Neurospora crassa (49), Let-502 kinase in Caenorhabditis elegans (50), p160 in platelets (51), Pk428 in heart and skeletal muscle (52), and Orb6 in fission yeast (28). DBF2, DBF20, Warts, Cot-1, Let-502, and Orb6 all play roles in cell division, size, morphology, or polarity. Thus, in general, DMPK may be of importance in regulation of cell cycle and cell volume through an interaction with PLM.

	ACKNOWLEDGEMENTS

We thank L. T. Horne and C. E. M. Gimmler for assistance.

	FOOTNOTES

^* This work was supported by the National Institutes of Health (to A. D. R., L. R. J., and J. R. M.), the Muscular Dystrophy Association (to J. G., A. D. R., and J. R. M.), the Piton Foundation (to A. D. R.), the American Heart Association Virginia Affiliate (to J. P. M.), an American Heart Association Established Investigator Award (to J. R. M.), and the long term support of the Clinical Research Unit, M01-RR-30, National Center for Research Resources, General Clinical Research Centers Program, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Box 6012, MR4 Bldg., UVAHSC, Charlottesville, VA 22908. Tel.: 804-982-3367; Fax: 804-982-3162; E-mail: pmounsey@virginia.edu.

Published, JBC Papers in Press, May 12, 2000, DOI 10.1074/jbc.M000899200

² J. P. Mounsey, D. Mistry, S. Reddy, and J. R. Moorman, submitted for publication.

³ J. P. Mounsey and J. R. Moorman, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: PKA, protein kinase A; PKC, protein kinase C; PLM, phospholemman; GST, glutathione S-transferase; DMPK, myotonic dystrophy protein kinase; AEBSF, the serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride; Tricine, N-[2- hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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