It has been shown that most I2 proteins are able to drastically decrease PP1c activity towards different promotion info non specific substrates such as Phosphorylase A and pNPP. As e pected, the addition of PfI2 in the nanomolar range significantly decreased PfPP1 activity up to 80%. To investigate the impact of KTISW and HYNE motifs on PfI2 regulatory activity we used deleted or mutated recombinant proteins. The contribu tion of the RV F motif is key to the function of PfI2 as both Nt deleted PfI2 and mutated PfI2 were unable to inhibit PfPP1 activity, whereas the involvement of the HYNE domain seems to be less important. Thus, although the PfI2W16A mutant is still able to bind to PfPP1, 12KTISW16 is a vital and a primary site for the inhibitory activity of PfI2.

To further evaluate the inhibitory activity of PfI2 and the role of the two motifs, we took advantage of the enopus model where oocytes are physiologically arrested in G2 M pro phase I. The injection of enopus I2 or anti PP1 antibodies into oocytes induced germi nal vesicle breakdown or GVBD. Plasmodium I2 is able to substitute for the enopus orthologue in this system since the microinjection of PfI2WT into oocytes promoted the progression to M phase, inducing GVBD and co immunoprecipitation e periments confirmed the interaction of PfI2 with enopus PP1c. This confirmed that PfI2 can function in cells without the need for the KGILK site and are in accordance with previous studies that showed the involvement of enopus I2 in the G2 M transition in acellular e tracts or the implication of Glc8 in the cell cycle.

Deletion, mutation or RNA interference studies carried out on inhibitor 2 have demonstrated its implication in the cell cycle, chromosome segregation and embryogenic deve lopment. In the case of PfI2, when deleted PfI2 lacking 12KTISW16 or mutated PfI2 were microinjected, no GVBD was observed, demonstrating the importance of both PfPP1 binding sites in the functional capacity of PfI2. Since the PfI2 mutated proteins are able to bind PP1 but unable to inhibit its function we sought to determine whether the pre injection of deleted or mutated PfI2 pro teins may block the role of wild PfI2. The pre injection of either PfI2 or PfI2W16A were able to block the induction of GVBD while PfI2Y103A did not. One e plan ation for these observations is that the HYNE dependent binding is critical as the injection of PfI2WT is able to dis place this mutated protein and to induce GVBD. When the HYNE site is not mutated the binding of PfI2 is suffi ciently stable to prevent its displacement. Closer e amination of the PfI2 peptide Brefeldin_A sequence revealed the presence of a consensus P TP motif, also present in other I2, in which the phosphorylation of the T within this site abrogated the function of I2.