REFERENTE DISCIPLINAR

It is well established that PKA mediates most of the actions of hormones that exert their effects by elevating the intracellular concentration of cAMP. PKA is implicated in numerous cell processes including:

Regulation of metabolism The most well-known function of PKA is in its regulation of glycogenolysis. Phosphorylation of phosphorylase kinase by PKA (Walsh et al, 1968) converts it to its active state in which it phosphorylates and activates its target protein, glycogen phosphorylase, making the latter enzyme more sensitive to its allosteric

activator, AMP. Glycogen phosphorylase catalyses the first step in the degradation of glycogen, and is dephosphorylated and inactivated by P P l, which is inhibited by PKA. Astrocytes store most of the brain’s glycogen. Glycogenolysis in astrocytes is elicited by vasoactive intestinal peptide, noradrenaline (NA) and adenosine, all of which elevate intracellular cAMP (Sorg and Magistretti, 1991). Similarly, the method by which adrenalin stimulates lipolysis in adipose tissue is via activation of PKA, which phosphorylates and activates HSL (Strâlfors and Belfrage, 1984). Astrocytes are the only brain cell population that oxidise fatty acids to ketone bodies (Edmond et al, 1987, Edmond, 1992, Staub et al, 1995). Cultured astrocytes produce ketone bodies at rates similar to those of hepatocytes (Blazquez etal, 1998, Guzman and Geelen, 1992, Guzman

et al, 1995). | The synthesis of these fatty acids is discussed on page 68.

Gene transcription The transcription factor CREB is a major nuclear substrate for PKA (Lalli and Sassone-Corsi, 1994), but is also phosphorylated on the same serine residue (ser-133) by MAPK in response to Ca^^-influx or growth factor activation (Pende et al,

1997). Phosphorylated CREB binds to a response element known as the cAMP- responsive element (CRE), in cAMP-inducible genes. This consists of an 8-base pair palindrome, and mediates the transcriptional induction of many genes including somatostatin, phosphoenolpyruvate carboxykinase (PEPCK), vasoactive intestinal peptide (VIP), enkephalin and synapsin I (Montminy, 1997). cAMP-induced gene expression is important in many cellular responses, including the establishment of long-term memory (Hunter, 1995), (or long-term potentiation, LTP). It is likely that different forms of LTP employ different isoforms of the PKA R subunit (Woo et al, 2000). In astrocytes, PKA suppresses the induction of inducible nitric oxide synthase (Feinstein et al, 1993); whereas expression of the Na^-dependent glutamate transporters, GLUT-1, and to a lesser extent the expression of the glutamate/aspartate transporter, GLAST, in astrocytes is increased by activation of PKA (Schlag et al, 1998).

Signal transduction CNS substrates for PKA involved in signal transduction include ion channels and receptors. K^ channels in neurons are regulated either positively or

negatively by PKA phosphorylation, depending on their type (Greengard, 1987). Voltage gated Ca^"^ channel conductance is increased by PKA phosphorylation (Greengard, 1987). Phosphorylation of the nicotinic acetylcholine receptor by PKA increases its rate of desensitization and thereby modulates synaptic transmission postsynaptically (Huganir

et al, 1986). The P-adrenergic receptor (P-AR) mediates the stimulatory effects of

catecholamines on many tissues through activation of adenylate cyclase. Prolonged exposure to p-adrenergic agonists results in desensitization of its adenylate cyclase activity, caused by phosphorylation of the P-AR by the P-AR kinase (Stadel et al, 1983). These effects can be reproduced by exogenous cAMP, suggesting that desensitization is stimulated by PKA. The phosphorylation of synapsin I, which is localized to presynaptic nerve terminals, increases neurotransmitter release and thereby modulates synaptic transmission presynaptically. Synapsin 1 is a substrate of PKA (Hemmings etal, 1989). DARPP-32, the PPl inhibitor is also a substrate for PKA, phosphorylation by PKA activates DARPP-32, enabling it to inhibit PPl (Hemmings et al, 1984b).

Cell motilitv Phosphorylation of myosin light chain (MLC) causes smooth muscle contraction. The enzyme responsible for this phosphorylation is MLC kinase (MLCK), which is inactivated by phosphorylation by PKA. In this way, adrenergic stimulation of smooth muscle produces muscle relaxation via activation of PKA and its inactivation of MLCK (Pato et al, 1995, Vérin et al, 1998). MLCK has also been identified in many regions of rat brain, as well as in cultured astrocytes and cerebellar granule cells, where it may have motility-related functions (Edelman et al, 1992). Approximately 30 % of PKA activity found in brain cofractionates with neuronal microtubule-associated protein 2 (MAP-2) (Vallee et al, 1981, Theurkauf and Vallee, 1982, Lohmann et al, 1984). MAP-2 is a high molecular weight protein (~ 270 kDa) which co-purifies with brain microtubules, and is a major substrate of PKA (Sloboda et al, 1975, Vallee, 1980). In astrocytes PKA phosphorylates the intermediate filament proteins GFAP and vimentin, major components of the glial cell cytoskeleton (McCarthy et al, 1985, Mobley and Combs, 1992). The intermediate filament proteins assemble to form filaments and undergo dynamic reorganisation during mitosis or differentiation (Jones et al, 1985, Lim

et al, 1973). The phosphorylation and déphosphorylation of intermediate filament proteins seem to have a critical role in these processes (Inagaki et al, 1987, Evans, 1988, Nakamura et al, 1992, Matsuoka et al, 1992). Astrocyte stellation is induced by elevation of intracellular cAMP in cultured cells, they then resemble the process-bearing, differentiated astrocytes observed in vivo (Shapiro, 1973, Moonen etal, 1976, Narumi et al, 1978, Shain et al, 1987). This stellation appears to be initiated by P-adrenergic stimulation of cAMP production (Shain et al, 1992, Harrison and Mobley, 1991), and is independent of GFAP phosphorylation (Pollenz and McCarthy, 1986). Stellate astrocytes participate in the formation of a neuronal network by providing neurotrophic support (Furukawaera/, 1986, Hatten era/, 1988) or by guiding neuronal migration (Rakic, 1971, Mason et al, 1988).

Cell proliferation. The stimulation of cell proliferation by growth factors is mediated via MAPK activation. In some cell types, including astrocytes, PKA inhibits cell proliferation through its inhibition of MAPK activity (Graves etal, 1993, Cook and McCormick, 1993, Sevetson et al, 1993, Wu et al, 1993), and also by blocking the translocation of MAPK from the cytosol to the nucleus (Kurino et al, 1996). PKA appears to phosphorylate and inactivate the upstream kinases, Raf-1 and B-Raf (see Figure 1.1, Peraldi et al, 1995, Ramstad et al, 2000, Liebmann, 2001) However, cAMP is a positive intracellular signal for cell proliferation in many differentiated cells (Dumont et al, 1989).