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The association of type 1, type 2A and type 2B phosphatases with the human T lymphocyte plasma membrane.

ABSTRACT: Several putative plasma-membrane-associated components of the T-lymphocyte signal-transduction pathway are phosphorylated during the initial events of cellular activation. Little is known about the control of dephosphorylation of these components. We have shown by immunoblotting that the type 1 phosphatase, the type 2A phosphatase and type 2B phosphatase (calcineurin) are associated with the plasma membrane of normal human T lymphoblasts and the human T leukaemic cell line Jurkat 6. The type 1 phosphorylase phosphatase activity is present in a latent form which can be stimulated synergistically by deinhibitor and p-nitrophenyl phosphate. The PCSH form of the type 2A phosphatase appears to be the predominant oligomer in the plasma-membrane fraction. All three phosphatases can be extracted from membranes with Nonidet P40, but whereas the type 1c and type 2Ac phosphatases separate into the detergent-poor phase of Triton X-114, calcineurin separates into both detergent-rich and -poor phases. It is probable that one or more of these three plasma-membrane-associated phosphatases play regulatory roles in determining the phosphorylation state of membrane-bound proteins involved in human T-cell activation.

SUBMITTER: Alexander DR

PROVIDER: S-EPMC1135499 | biostudies-other | 1988 Dec

REPOSITORIES: biostudies-other

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Project description: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.

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The association of type 1, type 2A and type 2B phosphatases with the human T lymphocyte plasma membrane.

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