MARCO TEÓRICO

PP2A is thought to exist primarily as a trimeric enzyme of variable subunit composition, consisting of a catalytic ( C) subunit and two regulatory subunits (A and B), although dimers have been purified, which may suggest that the regulatory subunits can dissociate (Cohen, 1989). The 36 kDa catalytic subunit, PP2Ac, of which two 97-98 % identical mammalian isozymes, a and p, exist (Da Cruz e Silva and Cohen, 1987, Stone et al,

1987), is complexed with the 65 kDa A subunit and one of at least 15 distinct regulatory B subunits ranging in molecular weight from 54 kDa to 130 kDa (Healy et al, 1991, Mayer-Jaekel et al, 1991, Pallas et al, 1992, Hendrix et al, 1993). As with the catalytic subunit, two distinct isozymes of the A subunit (a and p) are present in mammalian tissues (Hemmings et al, 1990, Walter et al, 1989, 1990). The amino acid sequences of the human a- and p-isoforms are 87 % homologous. It is likely that the B subunits influence substrate specificity and subcellular localisation. For example, the 72 kDa B subunit contains a potential nuclear localisation signal in its primary sequence (Hendrix

et al, 1993), which may account for the presence of PP2A in the nucleus (Turowski et

al,). Several isoforms of each B subunit of PP2A exist (Khew-Goodall and Hemmings,

1988, Axton et al, 1990), and while most subunits are ubiquitously expressed, two isoforms of the 55 kDa B subunit, P and y are mainly found in neuronal tissue (Mayer- Jaekel and Hemmings, 1994, Zolnierowicz et al, 1994).

Various mechanisms of regulation of PP2A have been identified. The main means of long-term PP2A regulation is by the differential expression of regulatory B subunit proteins that control enzyme specificity and activity by formation of oligomeric

complexes with the common AC core (Mumby and Walter, 1993). While the expression of A and C in different tissues is coordinated, it does not match that of the B subunits (Mayer-Jaekel et al, 1992, Ruediger, 1991). Association of the core complex with different B subunits confers distinct enzymatic properties to the complexes, such as alterations in substrate specificity (Chen et al, 1989, Imaoka et al, 1983), substrate targeting (Kosik, 1993, Sontagera/, 1996), kinetic parameters (Kamibayashi era/, 1994), subcellular localisation (McCright et al, 1996), cellular function (Mawal-Dewan et al,

1994, Zhao et al, 1997) and specific activity (Kamibayashi et al, 1992).

As with P P l, reversible phosphorylation has an important role in the short-term regulation of PP2A. The catalytic subunit or the AC heterodimer is inactivated by phosphorylation by various receptor tyrosine kinases including pp60''’*^^ p56'‘"'', the epidermal growth factor receptor, and the insulin receptor in vitro (Chen et al, 1992), so the activity of PP2A can be modified by extracellular signals. The inhibition of PP2A by tyrosine phosphorylation is interesting since PP2A is thought to suppress cell growth (Felix et al,

1990, Lee et al, 1991, Picard et al, 1991). Phosphorylation and inactivation of the PP2A C subunit is enhanced by OA, suggesting that the protein is rapidly autodephosphorylated (Chen et al, 1992). The activation of MAPK is a key event in signalling by growth factors (Sturgill and Wu, 1991), prolonged activation of this enzyme is required to trigger mitogenesis (Pages et al, 1993, Cowley et al, 1994). MAPK and its activator, MAPK kinase (MEK) are both inhibited by dephosphorylation by PP2A (Alessi et al, 1995). In this way, PP2A opposes entry into the cell cycle, which may explain its inhibitor, OA being a tumour-promoter (Suganuma et al, 1988).

The role of PP2A in opposing cell division does not end at inhibition of MAPK, the enzyme has potential functions in controlling several aspects of the cell cycle. PP2A has an inhibitory effect on progression through the cell cycle from G2 to M-phase (mitosis). Entry into mitosis is controlled by the cyclin-dependent protein kinases, more specifically by the complex formed between cyclin B and p34‘^^‘"^ (Langan et al, 1989) which is regulated by reversible phosphorylation. Phosphorylation of p34^‘^‘^^ on thr-161 is required

for activity (Gould et al, 1991), this is a similar site to that required for activation of MAPK, (Cobb etal, 1991, Payne al, 1991), whereas phosphorylation of thr-15 andtyr- 14 is inhibitory (Lundgren et al, 1991, Parker et al, 1991,1992). Phosphorylation of thr- 161 is catalysed by a p34‘"‘*'^^-activating kinase, also known as CAK, and the pathway leading to thr-161 phosphorylation is inhibited by PP2A. Thr-161 is not directly dephosphorylated by PP2A (Lee et al, 1994). activation requires dephosphorylation of thr-15 and tyr-14 by a protein phosphatase, cdc25. cdc25 is only active in the phosphorylated state and is kept inactive by PP2A (Clarke et al, 1993).

Studies on the physiological function of PP2A were initially concentrated on its role in the regulation of metabolism; it inhibits gluconeogenesis and lipolysis and stimulates fatty acid synthesis (Cohen, 1989). The enzymes that control the rate of fatty acid and cholesterol synthesis and lipolysis, ie. ACC, hydroxy methyl-glutaryl-coenzyme A reductase (HMG-CoAR) and hormone-sensitive lipase (HSL) are all dephosphorylated by PP2A (Ingebritsen et al, 1983a, Gaussin et al, 1997, Olsson and Belfrage, 1987). These three enzymes are also phosphorylated and inactivated by the same protein kinase, AMPK (Hardie, 1992, Garton et al, 1989). Other reports have suggested a role for PP2A in synaptic transmission and RNA splicing (Sim, 1992, Walter and Mumby, 1993) and dephosphorylation of DARPP-32, the inhibitor of P P l on thr-34, the site at which phosphorylation is required for activity (Desdouits etal, 1995). Barnes etal (1995) found PP2A localised at the synapses of CNS neurons, where it may alter the functions of phosphoproteins involved in synaptic plasticity, and where it was shown to dephosphorylate autophosphorylated CaM kinase H. PP2A selectively dephosphorylates the soluble form of CaM kinase II rather than that associated with the postsynaptic density where it is a substrate for PPl (Strack et al, 1997a).