Abstract
Protein phosphatase 2A (PP2A) holoenzymes are present in most cell types, including cardiac myocytes, where they provide a large proportion of serine (Ser, S) and threonine (Thr, T) phosphatase activity. PP2A family members comprise a catalytic C subunit, a scaffold A subunit and a B-‐type subunit, which regulates subcellular targeting, substrate specificity and catalytic activity. Previous studies in our laboratory showed that in adult rat ventricular myocytes (ARVM) the subcellular localization of the regulatory B56α subunit is altered in response to isoprenaline (ISO) stimulation. This encouraged further studies of B56 subunits in β-‐adrenergic regulation of cardiac PP2A, which form the basis of this PhD project.Immunoblot analysis of ARVM samples revealed that in this cell system regulatory B56α, -‐ γ and -‐δ subunits are expressed at protein level. Protein expression of PP2A scaffold and catalytic subunits was also confirmed. The subcellular distribution and ISO-‐induced translocation of PP2A subunits was investigated by fractionation of ARVM. B56α was depleted from the myofilament/nuclear compartment and was enriched in the cytosolic/membrane compartment of ISO-‐stimulated myocytes. This was observed also for PP2A scaffold and catalytic subunits.
Potential ISO-‐induced phosphorylation of B56δ, which is phosphorylated by protein kinase A (PKA) at S573 in non-‐cardiac cells, was explored. Use of the Phos-‐tagTM SDS-‐PAGE system suggested that B56δ was phosphorylated in ARVM in response to ISO stimulation. In further studies, by using a phospho-‐site-‐specific antibody, increased phosphorylation of S573 was revealed. Studies with propranolol, a non-‐selective β-‐adrenergic receptor (β-‐AR) antagonist, confirmed that the response is mediated by β-‐ARs. Studies with CGP 20712A (a β1-‐selective antagonist) and ICI 118,551 (a β2-‐selective antagonist) indicated that the response is mediated primarily by β1-‐ARs. Studies with compounds that inhibit (H89 and PKI) or activate (N6-‐Benz-‐cAMP) PKA showed that the activity of PKA is necessary and sufficient for the response.
To explore the functional role of B56δ phosphorylation at S573, adenoviral vectors encoding GFP-‐tagged human B56δ in wild type (WT) or mutated form, in which S573 is replaced by non-‐phosphorylatable alanine (Ala, A), were constructed and post-‐infection protein expression profiles in ARVM were characterized. The studies confirmed that heterologous WT B56δ is phosphorylated at S573 in response to ISO stimulation and that heterologous S573A B56δ is not phosphorylated at this site. Overexpression of WT B56δ but not S573A B56δ appeared to amplify ISO-‐induced phosphorylation of some PKA substrates. Further studies are now necessary to identify the direct targets of B56δ-‐PP2A and the impact of altered B56δ phosphorylation, and in particular to determine the physiological role of B56δ phosphorylation at S573 in β-‐adrenergic regulation of cardiac function.
| Date of Award | 2016 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Metin Avkiran (Supervisor) & Philip Eaton (Supervisor) |