D2 and Cdk4. Additionally, sustained ERK activity seems to be important for phosphorylation and nuclear localization of CDK2, further supporting progression through the restriction point. PI3K Pathway Signaling p85, another SH2-domain-containing protein, binds activated RTKs and recruits PI3K (phosphoinositide-3-kinase), phosphorylating the phospholipid PIP2 to PIP3, leading to recruitment of Akt (via its PH-domain). In addition to other pro-growth and pro-survival functions, Akt inhibits glycogen synthase kinase-3β (GSK3β), thereby preventing GSK3β -mediated phosphorylation and subsequent degradation of cyclin D1 (see figure). Akt further regulates G1/S components by mTOR-mediated promotion of cyclin D1 translation, phosphorylation of the Cdk inhibitors p27kip1 (preventing its nuclear import) and p21Cip1 (decreasing stability), and inactivating phosphorylation of the transcription factor FOXO4 (which regulates p27 expression). Together, this stabilization of cyclin D1 and destabilization of Cdk inhibitors favors G1 and G1/S-Cdk activity. Anti-mitogen Signaling Anti-mitogens like the cytokine TGF-β inhibit progression through the restriction point, causing a G1 arrest. TGF-β signaling activates Smads, which complex with E2F4/5 to repress Myc expression and also associate with Miz1 to activate expression of the Cdk inhibitor p15INK4b to block cyclin D-Cdk complex formation and activity. Cells arrested with TGF-β also accumulate p21 and p27. Mechanism Overview As described above, signals from extracellular growth factors are transduced in a typical manner. Growth factor binds to receptors on the cell surface, and a variety of phosphorylation cascades result in Ca2+ uptake and protein phosphorylation. Phosphoprotein levels are counterbalanced by phosphatases. Ultimately, transcriptional activation of certain target genes occurs. Extracellular signaling must be maintained, and the cell must also have access to sufficient nutrient supplies to support rapid protein synthesis. Accumulation of cyclin D's are essential. Cyclin D-bound cdks 4 and 6 are activated by cdk-activating kinase and drive the cell towards the restriction point. Cyclin D, however has a high turnover rate (t1/2<25 min). It is because of this quick turnover rate that the cell is extremely sensitive to mitogenic signaling levels, which not only stimulate cyclin D production, but also help to stabilize cyclin D within the cell. In this way, cyclin D acts as a mitogenic signal sensor. Cdk inhibitors (CKI), such as the Ink4 proteins and p21, help to prevent improper cyclin-cdk activity. Active cyclin D-cdk complexes phosphorylate retinoblastoma protein (pRb) in the nucleus. Unphosphorylated Rb acts as an inhibitor of G1 by preventing E2F-mediated transcription. Once phosphorylated, E2F activates the transcription of cyclins E and A. Active cyclin E-cdk