The Catalytic Subunit of Phosphoinositide 3-Kinase: Requirements for Oncogenicity

Abstract

The retroviral oncogene p3k (v-p3k) of avian sarcoma virus 16 (ASV16) codes for the catalytic subunit of phosphoinositide (PI) 3-kinase, p110␣. The v-P3k protein is oncogenic in vivo and in vitro; its cellular counterpart, c-P3k, lacks oncogenicity. Fusion of viral Gag sequences to the amino terminus of c-P3k activates the transforming potential. Activation can also be achieved by the addition of a myristylation signal to the amino terminus or of a farnesylation signal to the carboxyl terminus of c-P3k. A mutated myristylation signal was equally effective; it also caused a strong increase in the kinase activity of P3k. Mutations that inactivate lipid kinase activity abolish oncogenicity. The transforming activity of P3k is correlated with the ability to induce activating phosphorylation in Akt. Point mutations and amino-terminal deletions recorded in v-P3k were shown to be irrelevant to the activation of oncogenic potential. Interactions of P3k with the regulatory subunit of PI 3-kinase, p85, or with Ras are not required for transformation. These results support the conclusion that the oncogenicity of P3k depends on constitutive lipid kinase activity. Akt is an important and probably essential downstream component of the oncogenic signal from P3k.

Phosphorylation by Akt disables the anti-oncogenic activity of YB-1

“…While wild-type YB-1 strongly binds to the beads, YB-1* has no affinity for the cap structure in agreement with a previous report (Bader et al, 2003). The phosphomimetic S99E shows reduced cap-binding activity when (Aoki et al, 1998(Aoki et al, , 2000Bader et al, 2003). RCAS(B) vectors encoding the YB-1 mutants S99A and S99E were generated by site-directed mutagenesis using RCAS(B)-FLAG-YB-1 as template and the mutagenesis kit from Stratagene (La Jolla, CA, USA).…”

“…To test for interference with oncogenic cellular transformation by the PI3K pathway, we infected these cells with RCAS virus encoding myristylated p110a in a serial dilution. p110a is the catalytic subunit of class I A PI 3-kinases and, when myristylated, constitutively activates the Akt signaling pathway and induces oncogenic cellular transformation of avian cells (Chang et al, 1997;Aoki et al, 2000Aoki et al, , 2001. Wild-type YB-1 inhibited PI3K-induced oncogenesis by >80% in agreement with previous observations (Bader et al, 2003;.…”

Oncogenic signaling of class I PI3K isoforms

“…Myristylation of chicken p110a increases its oncogenicity and causes constitutive activation of Akt (Aoki et al, 2000). Myristylation of p110b and p110g now rendered these proteins able to induce constitutive phosphorylation of Akt, S6K, 4E-BP1 and GSK3b (Figure 1).…”

“…Constitutive membrane localization of p110a by a myristylation signal or viral gag protein activates oncogenic capacity that is not apparent in the wild-type protein (Chang et al, 1997;Aoki et al, 2000). Adding a myristylation signal to wild-type p110b and p110g likewise increases the oncogenicity of these isoforms.…”

“…Inhibitors or DMSO were added at the appropriate concentration during the final 2 h in serum-free F-10 Ham's medium. Western blotting was performed as previously described by Aoki et al (2000) and Kang et al (2005Kang et al ( , 2006. Abbreviations 4E-BP, eukaryotic initiation factor 4E binding protein; Akt, cellular homolog of murine thymoma virus akt8 oncogene; ASV16, avian sarcoma virus 16; CEF, chicken embryo fibroblasts; eIF4E, eukaryotic initiation factor 4E; Erk, extracellular signal-regulated kinase; FOXO, forkhead box O transcription factor; GSK3b, glycogen synthase kinase-3-beta; MAPK, mitogen-activated protein kinase; MEK, mitogenactivated protein kinase kinase 1; p70S6K, ribosomal protein S6 kinase, 70 kDa, polypeptide 1; p90rsk, ribosomal protein S6 kinase, 90 kDa, polypeptide 1; PI3K, phosphatidylinositol 3-kinase; Raf, v-raf-leukemia viral oncogene 1; Ras, rat sarcoma viral oncogene; RBD, Ras-binding domain; S6, S6 ribosomal protein; TOR, target of rapamycin.…”