A Novel cAMP-Stimulated Pathway in Protein Phosphatase 2A Activation

Abstract

Elevated cAMP in NRK-52E and L6 cells causes a marked reduction in the phosphorylation of numerous phosphoproteins, as detected initially with phosphoserine-specific antibodies. Here, we show that elevation of cAMP in NRK cells by forskolin/3-isobutyl-1-methylxanthine (IBMX) treatment decreased phosphorylation of substrates for different protein kinases, pointing to a common protein phosphatase as a target for cAMP-dependent regulation. Forskolin/IBMX treatment completely dephosphorylated a selective protein phosphatase 2A (PP2A) substrate, elongation factor-2 (EF-2), at its Ca 2ϩ calmodulin-dependent kinase site, and decreased phosphorylation of substrates for cyclin-dependent kinases, including retinoblastoma (Rb) protein. As reported before, forskolin/IBMX also decreased phosphorylation of a protein kinase C substrate, the Na,K-ATPase. The cAMP-stimulated dephosphorylation was blocked by the protein phosphatases 1 (PP1) and PP2A inhibitor okadaic acid at concentrations selective for PP2A but was not blocked by tautomycin at concentrations selective for PP1. The data implicate PP2A as a cAMP-activated phosphatase. Contrary to expectation, we found evidence that cAMP-dependent activation of PP2A did not depend on protein kinase A (PKA). Pretreatment of cells with the PKA inhibitor H89 abolished PKA activity measured in cell extracts and significantly decreased cAMP-activated phosphorylation of a known PKA substrate, ARPP-19, in cells, but failed to block the cAMP-stimulated dephosphorylation of EF-2, Rb, and other proteins. This novel pathway of PP2A activation, acting on the time scale of minutes, represents yet another example of a cAMP-mediated, PKA-independent signaling mechanism. Because PP2A is active toward a variety of endogenous substrates, cAMP-stimulated dephosphorylation may have complicated the interpretation of many prior studies.

ReSETting PP2A tumour suppressor activity in blast crisis and imatinib-resistant chronic myelogenous leukaemia

“…Restoration of PP2A activity impairs BCR/ABL leukaemogenesis: therapeutic effects of the PP2A activator forskolin Consistent with the negative effects of PP2A on BCR/ABL expression and function, restoration of PP2A activity back to normal levels via SET downregulation, PP2Ac overexpression or treatment with the potent PP2A activator forskolin (Feschenko et al, 2002) or with 1,9-dideoxy-forskolin (forskolin derivative that lacks adenylate cyclase-cAMP- (Seamon and Daly, 1981) activating function) induces marked apoptosis, reduces proliferation, impairs colony formation, inhibits tumorigenesis and restores differentiation of patient-derived myeloid CML-BC CD34 þ cells and/or BCR/ ABL-transformed cell lines, regardless of their degree of sensitivity to imatinib . Notably, the imatinib-, AMN107-and dasatinib-resistant T315I BCR/ABL þ cells are also sensitive to PP2A activation .…”

“…forskolin; Feschenko et al, 2002) or by interfering with the SET/PP2A interplay (i.e. PP2Ac overexpression, SET knockdown), promotes BCR/ABL tyrosine dephosphorylation (inactivation) which, in turn, trigger its proteasome-dependent degradation .…”

“…Although the molecular mechanism underlying PP2A activation by forskolin (Feschenko et al, 2002) in BCR/ABL-transformed cells is still largely unclear, it seems that it does not depend on increased cAMP or PKA activation .…”

p38 Mitogen-Activated Protein Kinase-γ Inhibition by Long-Acting β2Adrenergic Agonists Reversed Steroid Insensitivity in Severe Asthma

“…The phosphatase that specifically dephosphorylates p38 MAPK-␥ has not yet been identified. However, cAMP-PKA signals are reported to enhance activity of protein phosphatase (PP)2A (Feschenko et al, 2002). PP2C is also known as cAMP-coupled phosphatase (Yokoyama et al, 1995), and PP1 and PP5 Fig.…”

FSH-induced phosphoprotein phosphatase 2A-mediated deactivation of particulate phosphodiesterase-4 activities is abolished after alteration in proteoglycan synthesis in immature rat Sertoli cells

“…The precise mechanisms by which FSH itself limits excessive cAMP catabolism by reversing phosphorylation of particulate PDE4 activities are still unknown. Previous studies have shown that cAMP transiently up-or down-regulates PP2A activity in various cell types (Feschenko et al 2002, Moon & Lerner 2003, Pullar et al 2006, but the regulatory mechanisms controlling PP2A activity are not fully understood. In Sertoli cells, increase in PP2A activities under FSH conditions does not result from transcriptional up-regulation of PP2A-C isoforms, in agreement with the autoregulatory mechanism preventing fluctuation in PP2A-C translation (Baharians & Schonthal 1998).…”