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*
- § To whom correspondence should be addressed: Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-3635; Fax: 215-728-2741.
Abstract
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.
Footnotes
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↵‡ Supported in part by the Scientific Exchange Program of the National Cancer Institute and the Consiglio Nazionale delle Ricerche-Italy.
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↵* This work was supported in part by United States Public Health Service Grant CA57436. Additional support was provided by United States Public Health Service Grant CA06927 and by an appropriation from the Commonwealth of Pennsylvania to the Fox Chase Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- PH
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pleckstrin homology
- PI3-K
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phosphatidylinositol 3-kinase
- PDGF
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platelet-derived growth factor
- PPI
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phosphorylated phosphoinositides
- PDGFRβ
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platelet-derived growth factor receptor β
- D-PBS
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Dulbecco's phosphate-buffered saline
- HA
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hemagglutinin
- DMEM
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Dulbecco's modified Eagle's medium.
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↵2 A. Bellacosa and P. N. Tsichlis, unpublished data.
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↵3 N. N. Ahmed, T. O. Chan, A. Bellacosa, and P. N. Tsichlis manuscript in preparation.
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↵4 J. D. Ceci, C. Patriotis, A. M. Makris, C. Tsatsanis, R. Kovatch, D. A. Swins, N. A. Jenkins, P. N. Tsichlis, and N. G. Copeland, manuscript in preparation.
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- Received July 26, 1996.
- Revision received September 17, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
