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Figure 17.2 ClustalW sequence alignment of N-terminal domains of DdPI3K1,
Dictyostelium PI3K1 (GenBank accession #P54673); DdPI3K2, Dictyostelium PI3K2
(accession #P54674); and HsPI3Kg, human PI3Kg (accession #P48736). The N-terminal
domain of PI3K1 starts from 139 and ends at 271. This is the minimum region responsible
for the translocation of PI3K1 (S. Funamoto and R. A. Firtel, unpublished observation)
but slightly conserved between PI3K1, PI3K2, and p110g (Figure 17.2). The
N-terminal region translocates in pi3k1/2 knockout cells and wild-type cells
treated with LY294002, suggesting that PI3K translocation is independent of
its kinase activity. Expression of the N-terminal deleted mutant of PI3K1 (D2-
492) failed to complement the phenotype of the pi3k1/2 knockout mutant. By
co-expression of PI3K1-CFP with PhdA-YFP as a D3-PI probe in living cells,
it was found that PI3K1 translocates with kinetics similar to those of PH
domain translocation (Funamoto et al., 2002). The data suggest that targeting
of PI3Ks to the membrane by the novel N-terminal domain is one of the
important steps for its activation.
Constitutive localization of PI3K1 on the membrane through the use of
an N-terminal myristoyl tag from Src did not cause constitutive activation
of downstream PI3K1 effector pathways, suggesting that PI3K localization
and activation are independent events. A point mutation in the PI3K1
Ras-binding domain blocked PI3K activation, suggesting that PI3K
interaction with an active Ras protein through the Ras-binding domain
is a key event to activate PI3K1 localized on the membrane (Pacold et al.,
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