Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.

Abstract

Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N-and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.The transforming proteins of small DNA tumor viruses form multiple complexes with cellular proteins involved in signal transduction and growth control. These interactions play an important role in virus-induced tumorigenesis. Simian virus 40 (SV40) large T binds to the tumor suppressor proteins p53 (40, 42) and Rb (16) and presumably inactivates their function. Polyomavirus medium T associates with pp6Oc-src and activates its protein-tyrosine kinase activity (4,12,14). It also binds to other members of the c-src family (9,36,39). In addition, medium T binds to and activates phosphatidylinositol-3 kinase (13, 32). Moreover, medium T forms a complex with two cellular proteins of approximately 61 and 37 kDa (23,24,35,50,59,60,64). The two proteins are associated with each other in uninfected cells (23). The medium T antigen of nontransforming hrt mutants (3) does not form a complex with the 61-kDa and 37-kDa proteins but binds to the 73-kDa heat shock protein instead (23,24,35,50,60,64,70). These data suggest that complex formation between medium T and the 61-kDa and 37-kDa proteins might be necessary for transformation. SV40 small T forms a complex with two cellular proteins of approximately 56 and 32 kDa (76). These proteins are also associated with polyomavirus and BK virus small T (55, 56). They are identical to the medium-T-associated 61-kDa and 37-kDa proteins, respectively (50, 71).The 61-kDa protein was purified and partially sequenced, and its cDNA was cloned from a human cDNA library (72). Its predicted amino acid sequence revealed a protein consisting of 15 imperfect repeats, most of which are 39 amino acids long. It had no resemblance to known proteins in data banks. The 37-kDa protein was also purified, partially se-* Corresponding author. t Present address:

Reduced expression of the A? subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the A? and A? subunit genes

“…22 Importantly, we have shown that in every case the mutation renders the A␣ subunit defective in binding B or C subunits, depending on whether the mutation is located in the N-terminal B subunit binding domain (repeats 1-10) or in the C-terminal C subunit binding region (repeats [11][12][13][14][15] (Fig. 1).…”

“…B subunits bind to repeats 1-10 and the C subunit to repeats 11-15. 15,16 B and C subunits physically interact while bound to A␣ (Fig. 1).…”

“…[11][12][13][14] A model demonstrating how the A, B and C subunits interact in the holoenzyme is shown in Figure 1. 15,16 The existence of so many regulatory subunits suggests that the various forms of PP2A have distinct functions. In addition, numerous interactions of PP2A with growth regulatory viral [17][18][19] and cellular 20 proteins have been reported, underscoring its regulatory role.…”

Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation

“…Water-treated and excysting cysts (in Stages 1 and 2 and 30 and 60 min excystation) were dried on the coverslips. Subsequently, cytoskeletons, permeabilized trophozoites and cysts were blocked for 1 h in blocking buffer (5% goat serum, 1% glycerol, 0.1% bovine serum albumin, 0.1% fish gelatin and 0.04% sodium azide) and incubated for 1 h with the (nonmethylation sensitive) rabbit anti-PP2A-C polyclonal antibody directed against a synthetic peptide (KVTRRTPDYFL) corresponding to the C terminus of the human PP2A-C subunit [25] or the mouse monoclonal antibody against CWP-1 [26]. As a control for specificity, the Giardia C-terminal PP2A-C peptide 'HISRRVPDYFL' (GenScript Corporation) was preincubated for 30 min with the PP2A-C antibody.…”

“…For detection with the methylation sensitive antibody, filters were treated for 10 min with 0.2 M KOH prior to blocking of the membrane; control membranes were untreated. Filters were incubated with either the methylation sensitive (Ab 299/309 ) [29] or the non-methylation sensitive anti-PP2A-C polyclonal antibody [25], the rabbit polyclonal antibody against PDI-2 [30] as a protein loading control or the mouse monoclonal antibody against CWP-1. As a control for specificity, the KVTRRTPDYFL peptide used for immunization [25] or the Giardia C-terminal PP2A-C peptide HISRRVPDYFL (GenScript Corporation) was pre-incubated with the non-methylation sensitive PP2A-C antibody for 30 min.…”