Anti-PPP2CA monoclonal antibody, clone 2.U.28

Type 2A protein phosphatases (PP2As) are highly expressed in the brain and regulate neuronal signaling by catalyzing phospho-Ser/Thr dephosphorylations in diverse substrates. PP2A holoenzymes comprise catalytic C-, scaffolding A-, and regulatory B-type subunits, which determine substrate specificity and physiological function. Interestingly, de novo mutations in genes encoding A- and B-type sub-units have recently been implicated in intellectual disability (ID) and developmental delay (DD). We now report 16 individuals with mild to profound ID and DD and a de novo mutation in PPP2CA, encoding the catalytic C alpha, subunit. Other frequently observed features were severe language delay (71%), hypotonia (69%), epilepsy (63%), and brain abnormalities such as ventriculomegaly and a small corpus callosum (67%). Behavioral problems, including autism spectrum disorders, were reported in 47% of individuals, and three individuals had a congenital heart defect. PPP2CA de novo mutations included a partial gene deletion, a frameshift, three nonsense mutations, a single amino acid duplication, a recurrent mutation, and eight non-recurrent missense mutations. Functional studies showed complete PP2A dysfunction in four individuals with seemingly milder ID, hinting at haploinsufficiency. Ten other individuals showed mutation-specific biochemical distortions, including poor expression, altered binding to the A subunit and specific B-type subunits, and impaired phosphatase activity and C-terminal methylation. Four were suspected to have a dominant-negative mechanism, which correlated with severe ID. Two missense variants affecting the same residue largely behaved as wild-type in our functional assays. Overall, we found that pathogenic PPP2CA variants impair PP2A-1356(delta) functionality, suggesting that PP2A-related neurodevelopmental disorders constitute functionally converging ID syndromes.

Cardiac hypertrophy occurs in response to increased hemodynamic demand and can progress to heart failure. Identifying the key regulators of this process is clinically important. Though it is thought that the phosphorylation of histone deacetylase (HDAC) 2 plays a crucial role in the development of pathological cardiac hypertrophy, the detailed mechanism by which this occurs remains unclear. Here, we performed immunoprecipitation and peptide pull-down assays to characterize the functional complex of HDAC2. Protein phosphatase (PP) 2 A was confirmed as a binding partner of HDAC2. PPP2CA, the catalytic subunit of PP2A, bound to HDAC2 and prevented its phosphorylation. Transient overexpression of PPP2CA specifically regulated both the phosphorylation of HDAC2 S394 and hypertrophyassociated HDAC2 activation. HDAC2 S394 phosphorylation was increased in a dose-dependent manner by PP2A inhibitors. Hypertrophic stresses, such as phenylephrine in vitro or pressure overload in vivo, caused PPP2CA to dissociate from HDAC2. Forced expression of PPP2CA negatively regulated the hypertrophic response, but PP2A inhibitors provoked hypertrophy. Adenoviral delivery of a phosphomimic HDAC2 mutant, adenovirus HDAC2 S394E, successfully blocked the anti-hypertrophic effect of adenovirus-PPP2CA, implicating HDAC2 S394 phosphorylation as a critical event for the anti-hypertrophic response. PPP2CA transgenic mice were protected against isoproterenolinduced cardiac hypertrophy and subsequent cardiac fibrosis, whereas simultaneous expression of HDAC2 S394E in the heart did induce hypertrophy. Taken together, our results suggest that PP2A is a critical regulator of HDAC2 activity and pathological cardiac hypertrophy and is a promising target for future therapeutic interventions.