The PTEN tumor suppressor gene encodes a phosphatidylinositol 3P P-phosphatase that is inactivated in a high percentage of human tumors, particularly glioblastoma, melanoma, and prostate and endometrial carcinoma. Previous studies showed that PTEN is a seryl phosphoprotein and a substrate of protein kinase CK2 (CK2). However, the sites in PTEN that are phosphorylated in vivo have not been identi¢ed directly, nor has the e¡ect of phosphorylation on PTEN catalytic activity been reported. We used mass spectrometric methods to identify Ser 370 and Ser 385 as in vivo phosphorylation sites of PTEN. These sites also are phosphorylated by CK2 in vitro, and phosphorylation inhibits PTEN activity towards its substrate, PIP3. We also identify a novel in vivo phosphorylation site, Thr 366 . Following transient over-expression, a fraction of CK2 and PTEN co-immunoprecipitate. Moreover, pharmacological inhibition of CK2 activity leads to decreased Akt activation in PTEN+/+ but not PTEN3 3/3 3 ¢broblasts. Our results contrast with previous assignments of PTEN phosphorylation sites based solely on mutagenesis approaches, suggest that CK2 is a physiologically relevant PTEN kinase, and raise the possibility that CK2-mediated inhibition of PTEN plays a role in oncogenesis. ß

H-Ras, K-Ras, and N-Ras regulate cellular growth and survival and are often activated by somatic mutation in human tumors. Although oncogenic lesions occur in a single Ras isoform within individual tumors, it is unclear whether the remaining wild-type isoforms play supporting roles in tumor growth. Here, we show that oncogenic and wild-type Ras isoforms play independent and nonredundant roles within the cell. Oncogenic Ras regulates basal effector pathway signaling, whereas wild-type Ras mediates signaling downstream of activated receptor tyrosine kinases (RTK). We show that both are necessary for exponential growth of Ras-mutant cell lines. Furthermore, we show that oncogenic Ras desensitizes signaling from EGF receptor (EGFR). Depletion of oncogenic Ras with siRNA oligonucleotides relieves this negative feedback, leading to the hyperactivation of EGFR and wild-type Ras signaling. Consistent with this model, combining oncogenic Ras depletion with EGFR inhibition potently increases cell death. SIGNIFICANCE:The results of this study highlight a novel role for wild-type Ras signaling in cancer cells harboring oncogenic RAS mutations. Furthermore, these fi ndings reveal that therapeutically targeting oncogenic Ras signaling alone may be ineffective owing to feedback activation of RTKs, and suggest that blocking upstream RTKs in combination with downstream effector pathways may be benefi cial in the treatment of Ras-mutant tumors. Cancer Discov; 3(1);

Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2␣ and/or CK2␣) and two CK2␤ regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2␤ gene is required for early embryonic development, while the CK2␣ subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2␣ gene in the mouse germ line. Embryos lacking CK2␣ have a marked reduction in CK2 activity in spite of the presence of the CK2␣ subunit. CK2␣ ؊/؊ embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2␣ appears to play an essential and uncompensated role in mammalian development.