The inhibition of rabbit lens epithelial cell proliferation induced by insulin, IGF-1, and FGF-2 by PI-3K inhibitors indicates that PI-3K activation could be important during cell proliferation. Therefore, we examined the effects of these growth factors on PI-3K activation. Quiescent rabbit lens epithelial cell cultures were stimulated with insulin, IGF-1, FGF-2, and PDGF for 10 minutes. Because growth factor binding triggers the autophosphorylation of the receptor tyrosine kinase domain and association of activated PI-3K with receptor,
22 26 27 the stimulated lens epithelial cell extracts were immunoprecipitated with anti-PY antibody and the PI-3K activity associated with these immunoprecipitates was determined
(Fig. 2) . Consistent with our previous findings in bovine lens epithelial cells,
22 both insulin and IGF-1 stimulated PI-3K activation. Insulin and IGF-1 activated PI-3K by approximately 200% to 300% when compared with the untreated control. However, treatment of cells with FGF-2 had little or no effect on PI-3K activation. Although PDGF did not promote any significant proliferation, it stimulated PI-3K activity by more than 300%. Next, we examined the activation of Akt, an important downstream kinase of the PI-3K-signaling pathway. This kinase plays a prominent role in cell proliferation and prevention of apoptosis.
21 Activation of Akt is necessary for cell-cycle progression.
28 29 Phosphorylation of certain serine residues of this protein (ser308 and ser473) by upstream PI-3K-dependent kinases (PDKs) stimulate Akt.
21 We determined Akt activation after stimulating rabbit lens epithelial cells for 10 minutes with growth factors, by Western immunoblot, using a phospho-specific Akt antibody. Insulin and IGF-1 caused an eight- to ninefold increase in the levels of phospho-Akt (pAkt;
Figs. 3A 3B ) compared with untreated control cultures. Consistent with its effect on PI-3K stimulation, FGF-2 induced very little activation of Akt. However, PDGF (50 ng/mL), which stimulated PI-3K significantly, as did insulin (200 nM) and IGF-1 (50 nM), was not as effective as insulin or IGF-1 in activating Akt. A twofold increase in Akt phosphorylation was observed in the presence of PDGF. Increased pAkt levels were also found in proliferating cells cultured in the presence of IGF-1 for 48 hours
(Fig. 3C) . The presence of wortmannin or LY294002 in proliferating cultures inhibited the IGF-1-induced activation of Akt, indicating that phosphorylation of Akt is dependent on PI-3K activation and that these compounds actually inhibited PI-3K activity throughout the entire assay period (up to 48 hours). It should be noted that in cultures treated with wortmannin, IGF-1-induced Akt activation was not completely blocked, perhaps because of the loss of inhibitory potency of wortmannin, as this compound is unstable in aqueous medium over a prolonged period. The changes in the levels of pAkt observed in the presence of IGF-1 and PI-3K inhibitors over a 48-hour period were not due to changes in the expression of the enzyme, because the amount of total Akt remained unchanged
(Fig. 3D) . These results suggest that activation of PI-3K/Akt could be important for rabbit lens epithelial cell proliferation promoted by insulin and IGF-1.