PP1γ functionally augments the alternative splicing of CaMKIIδ through interaction with ASF.

Protein phosphatase 1 (PP1) and Ca2+/calmodulin-dependent protein kinase δ (CaMKIIδ) are upregulated in heart disorders. Alternative splicing factor (ASF), a major splice factor for CaMKIIδ splicing, can be regulated by both protein kinase and phosphatase. Here we determine the role of PP1 isoforms in ASF-mediated splicing of CaMKIIδ in cells. We found that 1) PP1γ, but not α or ß isoform, enhanced the splicing of CaMKIIδ in HEK293T cells; 2) PP1γ promoted the function of ASF, evidenced by the existence of ASF-PP1γ association as well as the PP1γ overexpression- or silencing-mediated change in CaMKIIδ splicing in ASF-transfected HEK293T cells; 3) CaMKIIδ splicing was promoted by overexpression of PP1γ and impaired by application of PP1 inhibitor 1 (I1PP1) or pharmacological inhibitor tautomycetin in primary cardiomyocytes; 4) CaMKIIδ splicing and enhancement of ASF-PP1γ association induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) were potentiated by overexpression of PP1γ and suppressed by inhibition of PP1γ with I1PP1 or tautomycetin in primary cardiomyocytes; 5) functionally, overexpression and inhibition of PP1γ, respectively, potentiated or suppressed the apoptosis and Bax/Bcl-2 ratio, which were associated with the enhanced activity of CaMKII in OGD/R-stimulated cardiomyocytes; and 6) CaMKII was required for the OGD/R induced- and PP1γ exacerbated-apoptosis of cardiomyocytes, evidenced by a specific inhibitor of CaMKII KN93, but not its structural analog KN92, attenuating the apoptosis and Bax/Bcl-2 ratio in OGD/R and PP1γ-treated cells. In conclusion, our results show that PP1γ promotes the alternative splicing of CaMKIIδ through its interacting with ASF, exacerbating OGD/R-triggered apoptosis in primary cardiomyocytes.

Rescue of cardiac failing and remodelling by inhibition of protein phosphatase 1γ is associated with suppression of the alternative splicing factor-mediated splicing of Ca2+/calmodulin-dependent protein kinase δ.

Our previous studies showed that protein phosphatase 1γ (PP1γ) exacerbates cardiomyocyte apoptosis through promotion of Ca(2+)/calmodulin-dependent protein kinase δ (CaMKIIδ) splicing. Here we determine the role of PP1γ in abdominal aorta constriction-induced hypertrophy and remodelling in rat hearts. Systolic blood pressure and echocardiographic measurements were used to evaluate the model of cardiac hypertrophy. Sirius red staining and invasive haemodynamic/cardiac index measurements were used to evaluate the effects of PP1γ or inhibitor 1 of PP1 transfection. Western blot, reverse transcription polymerase chain reaction and co-immunoprecipitation were applied to investigate the molecular mechanisms. Transfection of PP1γ increased the value of the heart mass index, left ventricular mass index and cardiac fibrosis, and simultaneously decreased the value of maximal left ventricular pressure increase and decline rate, ejection fraction, fractional shortening, and left ventricular end-diastolic pressure, as well as left ventricular systolic pressure. Transfection of inhibitor 1 of PP1, however, showed opposite effects on the aforementioned indexes. Overexpression of PP1γ potentiated CaMKIIδC production and decreased CaMKIIδB production in the hypertrophic heart. In contrast, inhibition of PP1γ re-balanced the CaMKIIδ splicing. Furthermore, CaMKII activity was found to be augmented or attenuated by PP1γ overexpression or inhibition, respectively. Further mechanistic studies showed that abdominal aorta constriction stress specifically increased the association of alternative splicing factor with PP1γ, but not with PP1ß. Overexpression of PP1γ, but not inhibitor 1 of PP1, further potentiated this association. These results suggest that PP1γ alters the cardiac hypertrophy and remodelling likely through promotion of the alternative splicing factor-mediated splicing of CaMKIIδ.

Micro(mi) RNA-34a targets protein phosphatase (PP)1γ to regulate DNA damage tolerance.

The DNA damage response (DDR) triggers widespread changes in gene expression, mediated partly by alterations in micro(mi) RNA levels, whose nature and significance remain uncertain. Here, we report that miR-34a, which is upregulated during the DDR, modulates the expression of protein phosphatase 1γ (PP1γ) to regulate cellular tolerance to DNA damage. Multiple bio-informatic algorithms predict that miR-34a targets the PP1CCC gene encoding PP1γ protein. Ionising radiation (IR) decreases cellular expression of PP1γ in a dose-dependent manner. An miR-34a-mimic reduces cellular PP1γ protein. Conversely, an miR-34a inhibitor antagonizes IR-induced decreases in PP1γ protein expression. A wild-type (but not mutant) miR-34a seed match sequence from the 3' untranslated region (UTR) of PP1CCC when transplanted to a luciferase reporter gene makes it responsive to an miR-34a-mimic. Thus, miR-34a upregulation during the DDR targets the 3' UTR of PP1CCC to decrease PP1γ protein expression. PP1γ is known to antagonize DDR signaling via the ataxia-telangiectasia-mutated (ATM) kinase. Interestingly, we find that cells exposed to DNA damage become more sensitive - in an miR-34a-dependent manner - to a second challenge with damage. Increased sensitivity to the second challenge is marked by enhanced phosphorylation of ATM and p53, increased γH2AX formation, and increased cell death. Increased sensitivity can be partly recapitulated by a miR-34a-mimic, or antagonized by an miR-34a-inhibitor. Thus, our findings suggest a model in which damage-induced miR-34a induction reduces PP1γ expression and enhances ATM signaling to decrease tolerance to repeated genotoxic challenges. This mechanism has implications for tumor suppression and the response of cancers to therapeutic radiation.