Tung O. Chan

The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase

The serine/threonine protein kinase encoded by the Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFR beta tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmannin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.

Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway

The BCR/ABL oncogenic tyrosine kinase activates phosphatidylinositol 3‐kinase (PI‐3k) by a mechanism that requires binding of BCR/ABL to p85, the regulatory subunit of PI‐3k, and an intact BCR/ABL SH2 domain. SH2 domain BCR/ABL mutants deficient in PI‐3k activation failed to stimulate Akt kinase, a recently identified PI‐3k downstream effector with oncogenic potential, but did activate p21 RAS and p70 S6 kinase. The PI‐3k/Akt pathway is essential for BCR/ABL leukemogenesis as indicated by experiments demonstrating that wortmannin, a PI‐3k specific inhibitor at low concentrations, suppressed BCR/ABL‐dependent colony formation of murine marrow cells, and that a kinase‐deficient Akt mutant with dominant‐negative activity inhibited BCR/ABL‐dependent transformation of murine bone marrow cells in vitro and suppressed leukemia development in SCID mice. In complementation assays using mouse marrow progenitor cells, the ability of transformation‐defective SH2 domain BCR/ABL mutants to induce growth factor‐independent colony formation and leukemia in SCID mice was markedly enhanced by expression of constitutively active Akt. In retrovirally infected mouse marrow cells, the BCR/ABL mutant lacking the SH2 domain was unable to upregulate the expression of c‐Myc and Bcl‐2; in contrast, expression of a constitutively active Akt mutant induced Bcl‐2 and c‐Myc expression, and stimulated the transcription activation function of c‐Myc. Together, these data demonstrate the requirement for the BCR/ABL SH2 domain in PI‐3k activation and document the essential role of the PI‐3k/Akt pathway in BCR/ABL leukemogenesis.

Phosphatidylinositol 3-Kinase Is Required for Integrin-Stimulated AKT and Raf-1/Mitogen-Activated Protein Kinase Pathway Activation

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.

Akt Is a Direct Target of the Phosphatidylinositol 3-Kinase ACTIVATION BY GROWTH FACTORS, v-src and v-Ha-ras, IN Sf9 AND MAMMALIAN CELLS

The Akt protooncogene encodes a serine-threonine protein kinase which is activated by growth factor-generated signals that are transduced via the phosphatidylinositol 3′-kinase (PI3-K). Earlier studies suggested that the activation of Akt by PI3-K may be mediated by the binding of D3-phosphorylated phosphoinositides to the Akt pleckstrin homology (PH) domain. On the basis of these studies, it was hypothesized that Akt is a direct PI3-K target. To test this hypothesis, we reconstituted the pathway of Akt activation in baculovirus-infected Sf9 cells. The results showed that Akt, which is normally catalytically inactive in these cells, was activated when coexpressed with the activated PI3-K. Moreover, they showed that activated forms of c-Ha-ras (v-Ha-ras) and c-src (v-src or srcY527F), two molecules that transduce growth factor-generated signals, also activate Akt in a PI3-K-dependent manner in Sf9 as well as NIH 3T3 cells. The activation of Akt by both growth factors and v-ras and v-src (or srcY527F) depends on the integrity of the Akt PH domain and carboxyl-terminal tail. These results show that Akt activation via the PI3-K can be faithfully reproduced in baculovirus-infected Sf9 cells. The same results support the hypothesis that Akt is a direct target of the PI3-K and identify cytoplasmic signaling molecules that may contribute to the transduction of PI3-K/Akt activation signals.

The Gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal.

The Gfi-1 proto-oncogene is activated by provirus insertion in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced thymomas and encodes a nuclear, sequence-specific DNA-binding protein. Here we show that Gfi-1 is a position- and orientation-independent active transcriptional repressor, whose activity depends on a 20-amino-acid N-terminal repressor domain, coincident with a nuclear localization motif. The sequence of the Gfi-1 repressor domain is related to the sequence of the repressor domain of Gfi-1B, a Gfi-1-related protein, and to sequences at the N termini of the insulinoma-associated protein, IA-1, the homeobox protein Gsh-1, and the vertebrate but not the Drosophila members of the Snail-Slug protein family (Snail/Gfi-1, SNAG domain). Although not functionally characterized, these SNAG-related sequences are also likely to mediate transcriptional repression. Therefore, the Gfi-1 SNAG domain may be the prototype of a novel family of evolutionarily conserved repressor domains that operate in multiple cell lineages. Gfi-1 overexpression in IL-2-dependent T-cell lines allows the cells to escape from the G1 arrest induced by IL-2 withdrawal. Since a single point mutation in the SNAG domain (P2A) inhibits both the Gfi-1-mediated transcriptional repression and the G1 arrest induced by IL-2 starvation, we conclude that the latter depends on the repressor activity of the SNAG domain. Induction of Gfi-1 may therefore contribute to T-cell activation and tumor progression by repressing the expression of genes that inhibit cellular proliferation.

AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.

PDK2: a complex tail in one Akt.

The kinase Akt contains two phosphatidylinositol-3 kinase (PI3K)-dependent phosphorylation sites, one in the activation loop (Thr308) and one in the carboxyl-terminal tail (Ser473), both of which are conserved among the members of the AGC kinase family. Under physiological conditions, the phosphorylation of Thr308 appears to be coordinately regulated with the phosphorylation of Ser473. Under experimental conditions, however, the two sites can be uncoupled, suggesting that their phosphorylation is controlled by different kinases and phosphatases. Phosphoinositide-dependent kinase 1 (PDK1), the kinase that phosphorylates the activation loop site, has been unambiguously identified. However, PDK2, a kinase that is hypothesized to phosphorylate the hydrophobic carboxyl-terminal site, remains elusive. This Perspective examines the regulation and biological significance of Akt phosphorylation at Ser473. The authors propose that Ser473 undergoes both autophosphorylation and phosphorylation by other kinases. Both events may be promoted by interactions between PDK1 and phosphorylated or phosphomimetically altered hydrophobic phosphorylation motifs in kinases associated with Akt. These interactions may induce conformational changes in Akt that make Ser473 accessible to phosphorylation.

Differential Roles of Akt, Rac, and Ral in R-Ras-Mediated Cellular Transformation, Adhesion, and Survival

ABSTRACT Multiple biological functions have been ascribed to the Ras-related G protein R-Ras. These include the ability to transform NIH 3T3 fibroblasts, the promotion of cell adhesion, and the regulation of apoptotic responses in hematopoietic cells. To investigate the signaling mechanisms responsible for these biological phenotypes, we compared three R-Ras effector loop mutants (S61, G63, and C66) for their relative biological and biochemical properties. While the S61 mutant retained the ability to cause transformation, both the G63 and the C66 mutants were defective in this biological activity. On the other hand, while both the S61 and the C66 mutants failed to promote cell adhesion and survival in 32D cells, the G63 mutant retained the ability to induce these biological activities. Thus, the ability of R-Ras to transform cells could be dissociated from its propensity to promote cell adhesion and survival. Although the transformation-competent S61 mutant bound preferentially to c-Raf, it only weakly stimulated the mitogen-activated protein kinase (MAPK) activity, and a dominant negative mutant of MEK did not significantly perturb R-Ras oncogenicity. Instead, a dominant negative mutant of phosphatidylinositol 3-kinase (PI3-K) drastically inhibited the oncogenic potential of R-Ras. Interestingly, the ability of the G63 mutant to induce cell adhesion and survival was closely associated with the PI3-K-dependent signaling cascades. To further delineate R-Ras downstream signaling events, we observed that while a dominant negative mutant of Akt/protein kinase inhibited the ability of R-Ras to promote cell survival, both dominant negative mutants of Rac and Ral suppressed cell adhesion stimulated by R-Ras. Thus, the biological actions of R-Ras are mediated by multiple effectors, with PI3-K-dependent signaling cascades being critical to its functions.