EL SENADO EN LA CONSTITUCIÓN DE 1845 1 Contexto histórico

Further experiments were designed in order to assess the contribution by CKII in the cell lysates. CKII is known to be regulated by extracellular signals and stimulation with EGF has been shown to activate CKII towards physiological substrates in EGF-treated A431 cells (Ackerman and Osheroff, 1989). Also, because CKII can use GTP as a phosphate donor (a property unique amongst the protein kinases), an excess of unlabelled GTP was used to quench the CKII signal. The results were surprising: Ptdlns 4KP and Ptdlns 4KpI were basally phosphorylated by soluble protein preparations of serum-starved A431 cells (p > pi). Upon EGF stimulation an increase in Ptdlns 4Kp and Ptdlns 4KpI phosphorylation is observed (increase in p » increase in pi). Quenching of the CKII signal with GTP revealed that most of the phosphorylation of Ptdlns 4Kp and Ptdlns 4KpI was due to CKII and that Ptdlns 4KP was also phosphorylated by an unknown kinase in an EGF-dependent manner. These preliminary results suggest that Ptdlns 4KP and Ptdlns 4KpI are differentially phosphorylated in response to growth factors. As with phosphorylation by purified CKII in vitro, no significant activation of Ptdlns 4K activity was observed upon EGF-stimulation (results not shown). The GTP used to quench the CKII signal also has the potential to activate G-protein pathways with unknown effects on Ptdlns 4Kp phosphorylation; specific CKII inhibitors should be employed in future studies and it may be necessary to identifiy the phosphorylation sites. The ability of cell lysates to phosphorylate recombinant Ptdlns 4K(I and the fact that Ptdlns 4Kp and Ptdlns 4KpI both bound large amounts of kinase activities from cell lysates (data not shown) suggests that it may be possible to affinity purify the cognate protein kinases.

The cloning of the human Ptdlns 4KP defines a novel group of Ptdlns 4Ks with substantial homology to the yeast Pikl gene. Database searches have confirmed the existence of other metazoan Piklp homologues in the genomes of C. elegans and D. discoideum. A particularly striking feature of this subbranch of the Ptdlns 4Ks is a conserved domain organisation which distinguishes the Pikl- from STT4-related Ptdlns 4Ks, specifically by the presence LKH3 region positioned between the LKH2 and the kinase domain, LK Hl. Preliminary experiments suggest that Ptdlns 4Kp may be phosphorylated by CKII and other protein kinases. Further experiments will be nesessary to establish the regulatory importance in vivo.

7.0 D iscu ssion

7.1

Synopsis

This thesis has presented the cloning of two novel enzymes, PtdlnsP K I la and Ptdlns 4Kp. PtdlnsPK I la belongs to a subgroup of the PtdlnsPK family whose members may generate PtdIns(4,5)P2 iri vivo by phosphorylation of PtdIns(5)P (Itoh et a l, 1998; Rameh et a l, 1997). Like PtdlnsPK lia , Ptdlns 4KP phosphorylates the D-4 position of the inositol headgroup but the preferred substrate is Ptdlns. A combination of affinity labelling and site-directed mutatgenesis was used to gain insight into the structure and function of the PtdlnsPK I la catalytic core, and the data presented in Chapter 4 concluded that the PtdlnsPK family is structurally related to both protein and PI kinases. PtdlnsPK I la was expressed in insect cells and used to generate monoclonal antibodies; one of these, 7H8, was characterised and found to be effective in immunoprécipitation, ELISA, immunofluorescence and western blotting. The availability of a specific immunological probe such as this should be useful in contributing to our future understanding of the cellular function of the type II PtdlnsPKs and the significance of the putative PtdIns(4,5)P2 biosynthetic pathway via PtdIns(5)P.

Ptdlns 4KP was cloned with the aim of further defining the mammalian Ptdlns 4Ks which catalyse an important first step in the synthesis of phosphoinositides. Ptdlns 4Kp was found to have several features in common with the yeast Ptdlns 4K Piklp but failed to rescue the essential function of Piklp when overexpressed in p ik l mutants. A putative casein kinase II site was identified in a splice variant of Ptdlns 4Kp (named Ptdlns 4KpI) and recombinant casein kinase II was found to preferntially phosphorylate Ptdlns 4Kpi in vitro. In vitro kinase assays using lysates from EGF-treated A431 cells indicate that Ptdlns 4Kp and pi proteins are differentially phosphorylated by more than one protein kinase. These phosphorylation events caused only small increases in activity and their significance is unclear.

7.2

The Function of Ptdlns 4-kinases and PtdlnsP kinases

At the time of writing some 7 PI 3-kinase, 3 Ptdlns 4K, and 6 PtdlnsPK isoforms are known in mammals (Chapter 1). The rapid expansion of cDNA databases and the use of homology-based PGR strategies allowed the majority of these to be identified on the basis of similarity with known proteins. Consequently, it has proven far easier to clone homologues than to assign physiological function.

The study of Pl-kinase function is hampered by several basic problems: firstly, the large number of PI 3-K, Ptdlns 4K, and PtdlnsPK isoforms makes placing specific gene products in linear pathways extremely difficult. This is largely a consequence of the combinatorial complexity of the problem, but difficulties also arise from the absence of a comprehensive panel of isoform-specific reagents at present. It is also extremely difficult

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