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 --> 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.
Highlights
Phosphorylation of membrane proteins by protein kinases is an important mechanism for receptor-mediated signal transduction in the regulation of cellular function
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 ICl(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
Summary
Phospholemman (PLM), a major membrane substrate for phosphorylation by protein kinases A and C, induces Cl currents (ICl(PLM)) when expressed in Xenopus oocytes. Co-expression of DMPK reduced the expression of PLM in oocyte membranes, suggesting a possible mechanism for the observed reduction in ICl(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. 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. We subsequently measured the amplitudes and biophysical characteristics of Xenopus oocyte-expressed ICl(PLM) in the presence and absence of the mRNA-encoding DMPK. We measured the effect of DMPK co-expression on the level of PLM expression in oocyte membranes
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