The p110α and p110β catalytic subunits of phosphoinositide 3-kinase (PI3K) regulate cell cycle events. Marqués et al. found that NIH 3T3 cells overexpressing p110β, but not p110α, divided more rapidly than control cells, whereas cells expressing a catalytically inactive p110β point mutant (Lys805→Arg; K805R) divided more slowly than control cells or cells expressing a catalytically inactive p110α mutant. Specifically, S phase progression was accelerated in cells overexpressing p110β and slowed in cells expressing the K805R mutant, suggesting a role for p110β in DNA elongation. Individual DNA fibers were isolated from synchronously dividing NIH 3T3 cells treated with inhibitors specific for p110α (PIK75) or for p110β (TGX221) and subsequently incubated with bromodeoxyuridine (BrdU). In DNA fibers from TGX221-treated cells, the BrdU-labeled sections were shorter in length and closer together compared with those from PIK75-treated cells, implying that replication fork progression was impaired. Subcellular fractionation and immunofluorescent staining of endogenous proteins in NIH 3T3 and several other cell types revealed that p110β was located primarily in the nucleus, whereas p110α was mostly cytosolic. During DNA replication, DNA elongation requires the tethering of DNA polymerase δ (Polδ) to chromatin through its processivity factor PCNA (proliferating cell nuclear antigen). Expression of wild-type p110β, but not p110α, increased PCNA binding to chromatin and to Polδ, whereas manipulations expected to decrease p110β activity, such as TGX221 treatment, decreased these interactions. The authors showed that p110β activated nuclear Akt, which phosphorylates Thr145 of the cell cycle inhibitor p21Cip, a phosphorylation that disrupts the interaction between PCNA and p21Cip and allows PCNA to bind to chromatin. Conversely, the association of PCNA with p21Cip expression increased in cells transfected with the inactive p110β mutant or treated with TGX221. Intriguingly, shRNA transfection had a greater impact than p110β inhibition on slowing S phase progression or decreasing PCNA association with chromatin, leading the authors to propose that p110β has a kinase-independent function, perhaps as a protein scaffold, in DNA elongation.
M. Marqués, A. Kumar, A. M. Poveda, S. Zuluaga, C. Hernández, S. Jackson, P. Pasero, A. C. Carrera, Specific function of phosphoinositide 3-kinase beta in the control of DNA replication. Proc. Natl. Acad. Sci. U.S.A. 106, 7525–7530 (2009). [Abstract] [Full Text]