Abstract
The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers.
Highlights
Gene in mice causes late onset myopathy and cardiac abnormalities strikingly similar to those of DM1 patients,[7,8] where they constitute main causes of lethality.[9]
In accord with data from others,[12] DMPK was associated to the outer mitochondrial membrane (OMM), as it was fully digested by trypsin under conditions that only disrupt external mitochondrial markers without affecting either intermembrane space (AIF and Omi) or matrix (CyP-D) components (Figure 1c)
We investigated whether Src interacts with HK II/DMPK and found that (i) Src co-immunoprecipitates with HK II, (ii) the presence of DMPK strongly enhances the HK II/Src interaction and (iii) a fraction of Src associated with HK II and DMPK is in its active form (Figure 3a)
Summary
Gene in mice causes late onset myopathy and cardiac abnormalities strikingly similar to those of DM1 patients,[7,8] where they constitute main causes of lethality.[9]. The discovery of a small set of targets of DMPK enzymatic activity suggested its involvement in diverse biological processes, including: (i) splicing regulation, as DMPK phosphorylates the RNA CUG-binding protein. CUG-BP/hNab[50]; (ii) modulation of Cl À currents and of intracellular Ca2 þ homeostasis, after the identification of phospholemman and phospholamban as DMPK substrates; and (iii) cytoskeletal rearrangements and protein quality control, as DMPK interacts with myosin phosphatase-target subunit 1 and with chaperones such as MKBP/HSPB2 (reviewed in Kaliman et al10), aB-crystallin/HSPB5 and HSP25/HSPB1.11 DMPK involvement in specific biochemical pathways remains poorly understood. Muscle fibers from DMPK À / À mice display swollen mitochondria with an abnormal ultrastructural organization.[8]. Biological processes in which mitochondria have a pivotal role, such as regulation of Ca2 þ homeostasis and apoptosis, are altered by changes in DMPK expression. DMPK À / À cardiomyocytes display increased levels of intracellular Ca2 þ ,14 whereas the apoptotic process is affected when the
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