EL SENADO EN EL PROYECTO DE CONSTITUCIÓN DE 1856 1 Contexto histórico

In Chapter 4 of this thesis, a nucleotide-binding residue of PtdlnsPK I la was identified. The position of this residue, K145, was used to optimise alignments of PtdlnsPKs with the serine/threonine protein kinase PKA such that it was possible to identify four regions of local homology corresponding to subdomains I, II, VIb and VII of the protein kinases. This study established that although the PtdlnsPKs are the most divergent branch of the protein-PI 3/Ptdlns 4K superfamily, homologies can be identified which correspond to structures involved in catalysis. Chapter 4 provided evidence for this relationship, and allowed the construction of rationally designed, catalytically inactive mutants for functional studies. However, proof of the relationship could only be provided by the three-dimensional structure of a PtdlnsPK.

The crystal structure of bacterially expressed recombinant PtdlnsPK Up (an isoform which displays 77 % identity to the kinase domain of PtdlnsPK Ila ) has recently been solved at 3.0 Â resolution (Rao et a l, 1998) and is the first lipid kinase structure reported. As predicted in Chapter 4, an invariant lysine residue in PtdlnsPK I la (K145) corresponds to the ATP binding lysine residue (K72) of PKA and lies below the predicted PtdlnsPK P-loop in a central cleft which represents the ATP-binding pocket (Figure 4.8; Rao et a l, 1998). Rao et a l used similarities between the crystal structures of PtdlnsPK Up and PKA to produce a model of the PtdlnsPK catalytic core in much the same way as the identification of the nucleotide binding residue of PtsInsPK I la was used to predict the positions of the catalytic and Mg^+-binding loops in Chapter 4. However, contrary to this thesis, Rao et a l predicted that the PtdlnsPK catalytic loop (PKA subdomain VIb) was the invariant sequence MDYSL in the PtdlnsPKs (Figure 4.2), and the Mg^+-binding loop (PKA subdomain VII) was IID which is also invariant in the PtdlnsPKs. Aligning the primary structure of PtdlnsPKs with PKA so that the MDYSL and IID sequences map onto the catalytic and Mg^+-binding loops respectively, made it necessary to introduce three major gaps in the kinase core (Rao et a l, 1998), an alignment that would not have been statistically optimal in the absence of structural data. However, both the MDYSL and IID motifs are well conserved and fold into a central cleft which also contains the ATP-binding site (Rao et a l, 1998). PtdlnsPK Iip was not crystallised in the presence of ATP, divalent ions, PtdIns(5)P substrate or a competitive

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inhibitor such as PtdIns(4,5)P2- Thus it is difficult to determine unequivocally whether the side chains of the critical residues are positioned appropriately for catalysis, especially since binding substrates and cofactor may alter the conform ation o f the catalytic cleft such that crucial residues are positioned differently.

An im portant question still remains: how are the substrate-binding sites o f the PtdlnsPKs, in particular the type I isoforms, able to accomodate such an unusually wide range o f lipid headgroups? This question is only likely to be answered by a high resolution structure of a PtdlnsPK enzyme bound to different substrates, A more detailed understanding o f the structural differences between PI kinases that are specific for different lipid substrates may allow the design o f novel specific inhibitors for research and clinical applications.

An interesting feature of the PtdlnsPK IIP structure is that it is predicted to be a disc­ shaped homodimer that interacts with lipid bilayers via a flattened, positively charged face. Like the p isoform, PtdlnsPK I la was predicted to be dimeric using two-hybrid analysis and gel filtration (M. dos Santos, S. Minogue, and J. Hsuan unpublished data) raising the possibility that this is a general property o f type II PtdlnsPKs. The relatively large membrane interface of the PtdlnsPK Iip suggests the potential to form a stable interaction w ith membranes that could be further stabilised by interaction with a membrane protein, phosphorylation or some other posttranslational modification. This putative property of the type II PtdlnsPK dimer may explain the observation that type II PtdlnsPK activity is distributed between the cytosolic and membrane fractions (Bazenet

et a i, 1990; Jenkins et a l, 1994).

The PtdlnsPK Iip sequence that binds the T N F a receptor is conserved in other type II

PtdlnsPKs (Castellino et al., 1997) and maps to a cleft on the opposite face from the

membrane interface (Rao et at., 1998). This raises the possibility that all type II isoforms

can interact with cell surface receptors. Ptdlns 4- and PtdlnsPK activities are reported to

associate with a basic juxtamembrane region of the EGF receptor (Cochet et al., 1991),

however the T N F a receptor does not contain such a sequence and neither do members of the trans-membrane 4 superfamily o f proteins which were recently found to co-purify

with the type II Ptdlns 4K (Yauch et a l, 1998).

7.5

Conclusion

There are many unresolved questions related to the physiological roles of Ptdlns 4- and PtdlnsPK s and significant challenges remain. Not least is the question o f how extracellular signals regulate the biosynthesis and flux of PtdIns(4,5)P2 through PLC and PI 3-kinase pathways. To address this question it will be necessary to identify the isoforms involved and dissect the mechanisms that regulate the activities of the relevant Ptdlns- and PtdlnsPKs. O f particular interest is the identity o f possibly novel enzymes including the type II Ptdlns 4K which has been implicated in a large number o f PI

signalling systems (Section 1.5.1.a) and the EGFR-associated PtdlnsPK (Cochet et al.,

1991), which remains completely uncharacterised.

The m etabolism of PtdIns(4,5)P2 which underlies exocytosis represents another important cellular model for Ptdlns 4- and PtdlnsPK function. However, it is not known how the supply of PtdIns(4,5)P2 is regulated or what the precise function of this lipid is. W hile the ability to reconstitute exocytotsis from permeablised cells using cytosolic com ponents provides a basis for assaying recom binant proteins and inhibitors, com plim entary experim ents are required to ensure that the correct isozym es are identified.

The putative role of phosphoinositides in regulating the cytoskeleton has recieved

much attention since the finding that Rho regulates a PtdlnsPK activity (Chong et al.,

1994) and that expression of type I PtdlnsPK s in various cell types leads to dramatic

changes in the actin cytoskeleton (Ishihara et al., 1998; Shibasaki et al., 1997, and

Section 1.10.1). Despite a growing body of data supporting the involvem ent o f PtdlnsPKs (and phosphoinositides in general) in the regulation of the actin cytoskeleton, much remains to be determined regarding the signalling pathways and molecular events which lead to this phenomenom.

Finally, perhaps the greatest challenge to investigators is to understand the bewildering complexity of phosphoinositide signalling which results from the combined complexities o f multiple isoforms, compartmentation phenomena, the heterogeneity o f signalling pathways, and crosstalk between signalling pathways. Before we can achieve a better understanding o f these aspects of cellular PI function we will need to characterise the proteins implicated in the regulation of these pathways in different cell types and generate corresponding isoform-specific reagents.

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8.0 Appendix