Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy

Olga Eleftheriadou,Jonathan Cowan,Andrew K Snabaitis,Brian E Wadzinski,Richard J Heads,Asvi Francois,Ali Ryan,Michael R Longman,Michael J Shattock,Andrii Boguslavskyi

doi:10.1007/s00395-017-0625-2

Abstract

Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACβ > PP2ACα > PP4C > PP6C), NRVM (PP2ACβ > PP2ACα = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACα > PP2ACβ > PP6C > PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACα, PP2ACβ, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (γH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of γH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium.

Highlights

Serine/threonine protein phosphatases and kinases control the phosphorylation status of many substrates and, as a consequence, regulate their activity and/or cellular localisation
Data confirm that gene transcription of all type 2A protein phosphatases was active in H9c2 cardiomyocytes (Fig. 1a), neonatal rat ventricular myocytes (NRVMs) (Fig. 1b), and adult rat ventricular myocytes (ARVMs) (Fig. 1c), as mRNA was detected for all type 2A phosphatases
We show that ARVM have relatively low levels of PP4C mRNA expression and undetectable levels of PP4C protein expression as determined by standard western analysis

Summary

Introduction

Serine/threonine protein phosphatases and kinases control the phosphorylation status of many substrates and, as a consequence, regulate their activity and/or cellular localisation. The serine/threonine protein phosphatases comprise a relatively large family of enzymes that are ubiquitously expressed and can be further subdivided into PP1, PP2A, PP2B (calcineurin), PP2C, PP3, PP5, and PP7 families based on their sensitivity to inhibitors such as okadaic acid [15] and their metal ion-dependent activity [14, 33]. The scaffold A- and C-subunits are expressed in varying degrees as a and b isoforms [3, 27, 30]; the complexity of activity towards substrates is largely controlled by the association of the heterodimeric core enzyme with a non-catalytic regulatory/targeting B subunit to form the active heterotrimeric holoenzyme [36]. The architecture of the heterotrimeric PP2A complex can result in at least 96 distinct holoenzymes [37]

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