Here we demonstrate that expression of DMPK-CUG repeat RNA induces hyper-phosphorylation of CUGBP1, which results in increased protein half-life and steady state levels. CUGBP1 was demonstrated to be hyper-phosphorylated in four different experimental systems including COS M6 cells expressing DMPK-CUG960 RNA, DM1 cell cultures, DM1 tissues, and an inducible DM1 mouse model. We also demonstrated that CUGBP1 hyper-phosphorylation in response to transient expression of DMPK-CUG960 RNA and in DM1 cultures requires PKC activity and that activation of PKC using a phorbol ester causes CUGBP1 hyper-phosphorylation and significantly increases protein half-life. CUGBP1 was directly phosphorylated by both PKC α and βII isozymes in an in vitro kinase assay. PKCα/βII was found to be activated in cells expressing DMPK-CUG960 RNA, DM1 cell cultures, DM1 tissues, and the inducible DM1 mouse model. Furthermore, a time course of molecular events following induction of DMPK-CUG960 RNA in mouse heart demonstrated that PKCα/βII activation, CUGBP1 hyper-phosphorylation, and elevation of CUGBP1 steady state levels occurred within six hours following induction of RNA expression. These results are consistent with a cause-effect relationship between expression of DMPK-CUG960 RNA, CUGBP1 hyper-phosphorylation, and increased CUGBP1 steady state levels. The findings from this paper provides a model for DM1 in which DMPK-CUG960 RNA triggers a signaling event that leads to PKCα/βII activation. Increased PKCα/βII activity causes hyper-phosphorylation of CUGBP1, most likely by a direct phosphorylation event. This phosphorylation event prolongs the half-life and increases the steady state levels of CUGBP1. Elevated CUGBP1 activity potentially leads to abnormalities in adult heart tissue by altering splicing of pre-mRNA targets and translation of target mRNAs.
CUGBP1 was previously shown to be a phosphoprotein and the phosphorylation state was found to be altered in DM1 heart tissue (Roberts et al., 1997
). In this previous study, analysis on one dimensional gels indicated nuclear accumulation of a hypo-phosphorylated isoform in DM1 tissues (Roberts et al., 1997
). This differs from our results in which expression of DMPK-CUG960
mRNA induced hyper-phosphorylation exclusively of nuclear CUGBP1 in cultured cells. In addition, we observed hyper-phosphorylation of CUGBP1 in DM1 tissue samples, cultured DM1 cells as well as within 6 hours following induction of DMPK-CUG960
mRNA in heart tissues from an DM1 inducible mouse model (Wang et al., 2007
). The differences in CUGBP1 mobility assayed on a 1D gel in the previous study do not represent the phosphorylation events identified in this report by the 2D analysis because the phosphorylation changes induced by transient expression of DMPK-CUG960
mRNA that were observed by 2D gel analysis were not apparent on 1D gels (data not shown).
Identification of multiple putative PKC phosphorylation sites on CUGBP1 led us to investigate the role of PKC in CUGBP1 phosphorylation. The PKC pathway is involved in many cellular processes such as growth, apoptosis, and differentiation (Musashi et al., 2000
). There are twelve PKC isozymes, all of which are serine/threonine kinases. Activation of these isozymes requires subsequent phosphorylation, lipid binding and translocation to membranes (Musashi et al., 2000
). Using activators and inhibitors of the PKC pathway, we identified PKC as required for CUGBP1 hyper-phosphorylation. We also demonstrated that recombinant PKCα and βII directly phosphorylate His-CUGBP1 in an in vitro
kinase reaction. Several PKC isozymes translocate to the nucleus upon activation and regulate nuclear events such as transcription, splicing and mRNA stability via phosphorylation of nuclear targets such as lamin B and histone H1 (Fields et al., 1988
; Martelli et al., 2006
; Martelli et al., 2003
; Omri et al., 1987
). It is also possible that CUGBP1 is phosphorylated in the cytoplasm and is efficiently translocated to the nucleus.
We found that that CUGBP1 hyper-phosphorylation mediated both by expression of expanded CUG repeats and PKC activation by phorbol esters leads to increased protein half-life. This is consistent with a previous report showed that CUGBP1 half-life was increased in COS7 cells expressing 170 CUG repeats (Timchenko et al., 2001
). Interestingly, the neuron specific embryonic lethal abnormal vision (nELAV) proteins, RNA binding proteins that are structurally similar to CUGBP1, are phosphorylated by PKCα after PMA treatment. PKC mediated phosphorylation increased the steady state levels of these proteins in rat hippocampus (Pascale et al., 2005
). It is possible that PKCα-mediated phosphorylation of CUGBP1 and related proteins could be a common mechanism for regulation of protein levels.
A key molecular feature of DM is the expression of embryonic alternative splicing patterns in adult tissues. For the ClC1 and IR genes, the failure of the embryonic isoforms to fulfill the functional requirements of adult tissues results in myotonia and insulin resistance, respectively (Charlet-B. et al., 2002
; Mankodi et al., 2002
; Savkur et al., 2001
). It is of interest that the first two proteins identified as CUG repeat binding proteins, CUGBP1 and MBNL1, are antagonistic regulators of alternative splicing and that both proteins normally regulate alternative splicing transitions during development (Ho et al., 2004
; Ladd et al., 2005
; Lin et al., 2006
). Normal modulation of MBNL1 activity during development is due to a postnatal transition from predominantly cytoplasmic to predominantly nuclear localization in skeletal muscle (Lin et al., 2006
). CUGBP1 protein expression is tightly down-regulated during normal post-natal development of heart and skeletal muscle (Ladd et al., 2005
; Lin et al., 2006
). However, CUGBP1 mRNA levels do not exhibit a corresponding decrease suggesting that protein steady state levels are regulated at the level of translation or protein stability (Ladd et al., 2005
; Lin et al., 2006
). We show a strong correlation between CUGBP1 hyper-phosphorylation, elevated steady state levels of CUGBP1 protein, and PKCα/βII activation in early developmental stages as well as DM1 tissues. In support of a role for PKC in modulating CUGBP1 levels, PKCα and βII levels are down regulated during heart development (Hamplova et al., 2005
; Schreiber et al., 2001
). These results strongly support a model in which DMPK-CUG960
mRNA expression disrupts the normal developmental regulation of CUGBP1 steady state levels mediated via a PKC pathway. These findings provide potential therapy options to reduce abnormal CUGBP1 levels to ameliorate DM1 phenotype.