TRANSCONECTOR ÓPTICO (OXC)

1.7.3.1 Regulation by phosphorylation

Phosphorylation of the CaM-PDE enzymes was demonstrated in the early studies of CaM- PDE enzymes purified from bovine tissues. Sharma and co-workers (1980) showed that the bovine brain 5 8 kDa CaM-PDE (PDE 1 A) could be phosphorylated by c AMP-dependent protein kinase (PKA) but this phosphorylation did not appear to affect the catalytic activity of the enzyme in this study. Studies by Sharma and Wang (1985) on the bovine brain CaM- PDE showed that the 61 kDa enzyme (PDEl A2), and not the 63 kDa (PDEIB) enzyme, was phosphorylated by PKA and that this caused a decrease in the affinity of the enzyme for calmodulin. The phosphorylated enzyme could be dephosphorylated by the calmodulin- dependent phosphatase, calcineurin, which was followed by an increase in the enzyme affinity for calmodulin (Sharma and Wang, 1985). Moreover, the same workers later showed that the 63 kDa bovine brain CaM-PDE could be phosphorylated by a preparation of bovine brain in the presence of calcium ions (Sharma and Wang, 1986a). As with phosphorylation of the 61 kDa CaM-PDE, phosphorylated 63 kDa CaM-PDE also showed a reduced affinity for calmodulin. The enzyme involved in the phosphorylation of the 63 kDa CaM-PDE was proposed as being the autophosphorylated calcium and calmodulin- dependent protein kinase H (CaM-kinase II) which is particularly abundant in the brain

(Sharma and Wang, 1986a; Hashimoto et al, 1989). Bovine brain 63 kDa CaM-PDE is

thought to have multiple phosphorylation sites for CaMKI I since tryptic digestions carried out on the phosphorylated enzyme revealed two major and two minor phosphorylated peptide fragments (Hashimoto et al, 1989).

In general, phosphorylation of the CaM-PDEs is blocked by the binding o f calmodulin to the enzyme and phosphorylation of CaM-PDEs results in the reduction of the affinity of

the enzymes for calmodulin. Although different kinases are involved in the

phosphorylation process (Sharma and Wang, 1985; Sharma, 1991) all the CaM-PDEs can be dephosphorylated by the calmodulin-dependent phosphatase, calcineurin (Sharma and Wang, 1986a), which is accompanied by an increase in the affinity of the enzymes for calmodulin.

The consensus sequences for the two kinases involved in the phosphorylation o f PDEl

enzymes are RRXS and RXXS/P(T) (Payne et al, 1983; Pearson et al, 1985) for cAMP-

dependent protein kinase (PKA) and calmodulin-dependent protein kinase (CaMKII) respectively. There is a strong requirement for the presence of an arginine residue (R) at the -3 position to the serine residue. The presence o f a second arginine at -2 is usually a positive determinant for PKA (RRXS) and a relatively strong negative determinant for CaMKII (Soderling et al, 1986).

The phosphorylation of PDEl A l and PDEl A2 was demonstrated in the early studies on CaM-PDEs (Sharma, 1991; Sharma and Wang, 1985). Sonnenburg and co-workers (1995) identified a potential PKA recognition sequence (RGRS^®'‘) at the N-terminal portion of the bovine lung PD ElA l which is within the second calmodulin-binding domain of the enzyme. Florio and co-workers (1994) identified serine 120 as well as serine 138 in bovine brain PDE1A2 as being targets for phosphorylation by PKA (RFRS*^®). PDE IB 1 has two potential phosphorylation sites at the carboxy-terminal portion (RQPS'*®®; RAAS^®^) (Yu

et al, 1997). PDEIBI has been shown to be phosphorylated in vitro by CaMKII but the

location of these sites o f phosphorylation have not been identified (Sharma and Wang, 1986a). CaMKII is known to phosphorylate target serine residues located at the C-terminal portion of at least one of its other target proteins - a N a ^ e x c h a n g e r (Fliegel et a l, 1992). This exchanger was also found not to be a substrate for phosphorylation by PKA or PKC.

1.7.3.2 Regulation by proteolytic degradation

The PD ElA l enzymes have been shown to be regulated through proteolytic cleavage by

m-calpain (Kakkar et al, 1998). Calpains are Ca^^-dependent cysteine proteases that are

widely distributed in the animal kingdom, from mammals to invertebrates but have not been described in plants or bacteria (Murachi, 1983; Mykles and Skinner, 1986; Carafoli and Molinari, 1998). They are sensitive to the inhibitor calpastatin as well as leupeptin (Carafoli and Molinari, 1998). Calpains are thought to be major mediators for calcium signalling in many tissues including the brain, lung and the liver (Murachi, 1983 ; Croall and Demartino, 1991; Sorimachi et al, 1997; Carafoli and Molinari, 1998). Substrates of calpains include myelin basic protein and tubulin as well as enzymes such as myosin light

forms that exist, m-calpains and p-calpains, differ in their sensitivity to Ca^^ with the former requiring millimolar concentrations o f Ca^^ and the latter requiring micromolar Ca^^ concentrations for activation. Target proteins with sequences enriched with the residues proline (P), glutamate (E), serine (S) and threonine (T), known as PEST motifs, are thought to be the signal for proteolytic cleavage by calpains (Rogers et al, 1986). Calpains target many different proteins and include transcription factors (c-Jun, c-Fos and nuclear factor

kB) as well as plasma membrane Ca^^-ATPase (Hirai eta/., 1991; Molinari et a l, 1 9 9 5; Liu

et al, 1996). The strength of the PEST sequence is determined by the PEST score which

has a range of -45 (weak) to +50 (strong). Generally, a PEST score between -5 and 0 denotes a weak strength, a score > 0 denotes a possible PEST sequence and a value >5

indicates the strong presence o f a PEST sequence (Rogers et al, 1986).

Kakkar and co-workers (1999) used a PEST-FTND program to identify the presence of a PEST sequence in the bovine PDEl A2 enzyme sequenced by Sonnenburg and co-workers (1996). Their analysis revealed a PEST sequence with a score of 7.36 located at the amino terminal portion of the enzyme (Figure 1.17). Cleavage by calpains does not usually occur within the PEST motifs but at a distant site. Kakkar and co-workers (1998) have demonstrated that the proteolytic cleavage of PDE1A2 by m-calpain was at a site approximately 30 amino acid residues away and that this cleavage yielded an enzymatically active calmodulin-independent 45 kDa fragment. N-terminal sequence analysis o f the 45 kDa fragment, together with the fact that the activity of the fragment was no longer affected by Ca^^/CaM, indicated that the calpain cleavage occurred at residues located between 120 and 138 (Figure 1.17). Biochemical analysis o f the cleavage process showed that the presence of CaM did not interfere with substrate recognition by calpain, and

phosphorylation of PDE1A2 also did not affect the cleavage process. The exact

significance of this cleavage is not known but the proteolysis of PDE1A2 to a CaM- independent enzyme in neural tissues has been postulated as being important in decreasing cAMP levels in these tissues. In diseases such as Parkinson’s disease there is a significant decrease in the cAMP levels and since PDE1A2 has been shown to be co-locahsed with calpains in the same region as the dopamine receptors, it is possible that PDE1A2 plays a role in the decreased cAMP levels in this region. Kakkar and co-workers (2002) have recently reported the calpain cleavage of rat heart PD E lA l by both m- and p,-calpains during hypoxic injury to the heart. As with PDE1A2, a CaM-independent fragment was

generated following calpain cleavage. Since increased c AMP has been associated with cell death (Jiang et ai, 1996), it is possible that the generation of a CaM-independent species in damaged cells limit further cell damage or death.

RFRS V NH^ IVHVVQ AGIFVERMYRKS Calpain c le a v a g e site RLLDTDDELSIQSDDSVPSEVR PE S T s e q u e n c e PDE1A2 COOH

RF^S PKA phosphorylation site

Calmodulin binding domains in PDE1A2 Conserved catalytic domain

Figure 1.17 Schematic showing the PEST motif and m-calpain cleavage site in bovine