Catalytically Inactive Protein Phosphatase 2A Can Bind to Polyomavirus Middle Tumor Antigen and Support Complex Formation with pp60 ^{c- src}

Abstract

Interaction between the heterodimeric form of protein phosphatase 2A (PP2A) and polyomavirus middle T antigen (MT) is required for the subsequent assembly of a transformation-competent MT complex. To investigate the role of PP2A catalytic activity in MT complex formation, we undertook a mutational analysis of the PP2A 36-kDa catalytic C subunit. Several residues likely to be involved in the dephosphorylation mechanism were identified and mutated. The resultant catalytically inactive C subunit mutants were then analyzed for their ability to associate with a cellular (B subunit) or a viral (MT) B-type subunit. Strikingly, while all of the inactive mutants were severely impaired in their interaction with B subunit, most of these mutants formed complexes with polyomavirus MT. These findings indicate a potential role for these catalytically important residues in complex formation with cellular B subunit, but not in complex formation with MT. Transformation-competent MT is known to associate with, and modulate the activity of, several cellular proteins, including pp60c-src family kinases. To determine whether association of MT with an active PP2A A-C heterodimer is necessary for subsequent association with pp60c-src , catalytically inactive C subunits were examined for their ability to form complexes containing pp60c-src in MT-expressing cells. Two catalytically inactive C subunit mutants that efficiently formed complexes with MT also formed complexes that included an active pp60c-src kinase, demonstrating that PP2A activity is not essential in cis in MT complexes for subsequent pp60c-src association.

Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells

“…However, phosphatase activity assays on the immune complexes revealed that, whereas the specific activity of the native HA-PP2Ac (218 nmol [$#P]P i released\min per mg) was similar to that of the purified rabbit skeletal muscle enzyme (180 nmol [$#P]P i released\min per mg), there was a 9-fold and 22-fold reduction in specific activity for the mutants D88N (24 nmol [$#P]P i released\min per mg) and H118N (9.7 nmol [$#P]P i released\min per mg) respectively, using a phospho-kemptide substrate (Table 1). These results are in agreement with those of Ogris et al [17,18] who detected a dramatic decrease in PP2Ac activity towards phosphorylated histone H1 (2 % of wild-type) and phosphorylase (8 % of wildtype) caused by a H118Q substitution [17]. PP2Ac complexes with PR65\A in i o to form a core dimer [1,3].…”

“…Recently, PP2Ac mutants mutated at the general acid residue His"") or residues involved in metal-ion binding were expressed in NIH3T3 cells. Immunoprecipitated proteins had impaired phosphatase activity, revealing conservation of the PPP catalytic mechanism [17,18].…”

A PP2A active site mutant impedes growth and causes misregulation of native catalytic subunit expression

“…The dominant‐negative phenotype of the H118N mutant in yeast cells had suggested that the mutant C subunit might compete for regulatory A subunit binding [Lizotte et al, 1999]. Regulatory A subunit binding by an H118Q mutant was found to be variable [Ogris et al, 1999a,b]. Transiently expressed HA‐tagged H118N also interacts with the A subunit, although binding may be weaker [Prickett and Brautigan, 2004].…”

“…5D and Prickett and Brautigan 2006). Indeed, the H118Q mutant binds the PME‐1 methylesterase more stably than the wild‐type protein does [Ogris et al, 1999b]. Furthermore, PME‐1 co‐purifies with an inactive form of the wild‐type C subunit; the inactive C subunit can be reactivated by treatment with the PP2A phosphatase activator (PTPA) [Longin et al, 2004], a regulatory factor that plays a key role in controlling PP2A conformation and activity [Fellner et al, 2003; Chao et al, 2006; Jordens et al, 2006; Leulliot et al, 2006].…”

Molecular insight into the viral biology and clinical features of trichodysplasia spinulosa

“…The involvement of MT antigen in cell proliferation is unknown for TSPyV, but is well described for murine polyomavirus (PyV) . PyV MT antigen is capable of driving cell transformation, and its physical association with PP2A plays an essential role in this process . PyV MT antigen–PP2A binding forms a scaffold that mediates MT regulation of key protein kinases such as src, shc and phosphoinositide 3‐kinase .…”