CATEGORÍA 4: INFLUENCIA DEL CONTEXTO SOCIAL (ECONÓMICO, ACADÉMICO Y GEOGRÁFICO) EN LAS

(Figure 4.6).

The catalytic subunit of PKA has been extensively studied using site-directed mutagenesis and the crystal structure solved (Knighton et al., 1991). Several structural features of PKA which are conserved in other phosphotransferase enzymes, are also conserved in the PtdlnsPK family. Most significantly, crucial residues implicated in nucleotide binding and catalysis are amongst the most highly conserved. These are summarised in Figure 4.1 and Table 4.1.

No P-loop consensus was found in the PtdlnsPKs but when the sequence just upstream of K145 was analysed less stringently, a potential loop region with the PtdlnsPK consensus sequence GXSGSXF can be identified. The fact that this was not detected in the initial analysis of PtdlnsPK I la is reminiscent of the PI 3-kinases where P-loops are not recognisable (Zvelebil et al., 1996). Although the majority of nucleotide binding proteins and protein kinases conform to the GXXXXG consensus sequence (Saraste et al., 1990), several do not, indicating that variation in this structure is allowed. Whether or not the PtdlnsPK consensus sequence GXSGSXF represents a phosphate binding motif needs to be determined experimentally but it is reasonable to assume that this represents the P-loop because of its position close to K145 and the fact that this is well conserved in the PtdlnsPKs (see Figures 4.2 and 4.7).

One of the most highly conserved regions of homology in the PtdlnsPK family resembles the protein kinase subdomain II which, on the basis of FSBA-labelling experiments, contains the nucleotide-binding site in protein kinases (Zoller et al., 1981; Zoller and Taylor, 1979), PI 3-kinases (Wymann et al., 1996) and PtdlnsPK (this study) families. Importantly, this residue is absolutely conserved and even conservative mutations at this position lead to a loss of activity (Wymann et al., 1996). The high degree of conservation reflects the proposed role of this residue in anchoring the a and P- phosphates of the ATP molecule (Knighton et al., 1991; Zheng et al., 1993).

Subdomain VIb in protein-, PI 3- and Ptdlns 4Ks represents the catalytic loop containing the catalytic aspartic and asparagine residues in the consensus DXXXXN. Modification or mutation of either of these residues leads to an inactive protein (Buechler and Taylor, 1988). The corresponding sequence in PtdlnsPK I la (DLKGST) contains K213 which also gives an inactive protein when mutated to an alanine residue. Most protein serine/threonine kinases also contain a lysine residue at this position which is responsible for binding the y-phosphate, but protein tyrosine kinases contain either an arginine or an alanine residue and in the PI 3-kinase family this position is conservatively substituted to a histidine residue. It is noteworthy that no residue corresponding to N 171 in the highly conserved subdomain VIb can be identified in the PtdlnsPKs. In PI3/4- and protein kinases this asparagine residue lies in the DXXXXN consensus sequence of the catalytic loop and although not directly implicated in catalysis, this residue stabilises the loop by hydrogen bonding to the backbone carbonyl groups of D 166 (Knighton et al..

1991). The corresponding sequence in PtdlnsPK I la is DLKGST and it is not known if the threonine residue can hydrogen bond in a similar manner.

Subdomain VII containing the DFG sequence is present in PtdlnsPKs as the invariant DYS motif. The critical amino acid in this sequence is the aspartic acid residue as it is invariant in protein and PI kinases and as its mutation in PKA abolishes activity. It is thought that the invariant aspartic acid residue is involved in chelating the primary magnesium ion (Knighton et a l, 1991; Zheng et a l , 1993). The changes from phenylalanine to tyrosine and glycine to serine residues represent conservative changes which are also found in protein kinases such as PVPKl from Phaseolus vulgaris, which has the sequence DFD (Lawton et a l, 1989), and the (3-andrenergic receptor kinase, which has the sequence DLG (Benovic et a l, 1989).

Determining the position of the nucleotide binding residue has allowed a more detailed analysis of the subdomain organisation of the PtdlnsPK family and although the importance of other critical residues such as D211 and D273 has not been addressed in this study, these residues can now be aligned with the protein and PI-3 and Ptdlns 4Ks with greater confidence. The data presented here clarify the structural relationship between the PtdlnsPKs and the Pl/protein kinase superfamily and support the hypothesis that the PtdlnsPK family is related to other phosphotransferases (Yamamoto et a l, 1995). Furthermore, as virtually all the conserved residues implicated in catalytic function in protein kinases also appear to be present in PtdlnsPKs, a distinct phosphotransferase mechanism, as previously suggested (Boronenkov and Anderson, 1995; Loijens et a l, 1996) is unlikely. Therefore, despite considerable diversity the PtdlnsPKs and members of the protein/PI kinase family appear to have evolved similar structures and do not constitute a completely unrelated family as previously stated (Hunter, 1995). The ongoing effort of our lab to solve the crystal structure of PtdlnsPK I la should determine to what extent the PtdlnsPK family is related to other kinases by a common tertiary structure.

In addition to providing basic information on structure, the construction of kinase-dead mutants provides important reagents for the future study of the PtdlnsPK function. It is anticipated that subtlely mutated, kinase-defficient PtdlnsPKs may inhibit signalling processes by acting as dominant negative proteins. These may prove to be useful in determining the mechanism of regulation of PtdlnsPKs, in particular the type II enzymes which have been proposed to lie in a novel PtdIns(4,5)P% biosynthetic pathway (Rameh et a l, 1997).

C-terminal proline-rich domain

None of the PtdlnsPKs contains any identifiable molecular interaction motif (Section 3.2.1). Limited homology to the chaperonin TCP-1 has been noted in the PtdIns(3)P 5- kinase Fab Ip and the putative PtdlnsPK C.e ORFl (Hsuan et a l, 1998; Yamamoto et a l,

1995, also see Figure 1.6b), although the function of this region is unknown. We and others have noted the presence of two short proline-rich sequences between amino acids

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