The Synthetic Peptide RPRAATF Allows Specific Assay of Akt Activity in Cell Lysates

Bozinovski, Steven; Cristiano, Briony E.; Marmy-Conus, Nelly; Pearson, Richard B.

doi:10.1006/abio.2002.5659

Cited by 17 publications

(22 citation statements)

References 29 publications

(29 reference statements)

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“…In agreement with previous studies (22), Ն80% of activity in cell lysates of an exponentially growing B lymphoma cell line was sensitive to pretreatment of the cells with PI3K inhibitors (Fig. 3A), with activity being equally inhibited in total cell lysates and Akt immunoprecipitates (Fig.…”

Section: Resultssupporting

confidence: 93%

“…1A) uses a nonradioactive isotope dilution approach and MS to quantify protein kinase activity toward the RPRAATF peptide (24) (hereafter named ''Aktide''), a highly selective substrate of protein kinases, such as Akt and SGK, downstream of PI3K (22,(24)(25)(26), with Akt͞PKB being particularly active toward this peptide. Indeed, protein kinases downstream of PI3K [chiefly Akt͞PKB and SGK (22,(24)(25)(26)] phosphorylate Aktide with faster kinetics than do other protein kinases, thus making the contribution of nonspecific protein kinases to overall Aktide kinase activity in cell lysates negligible (see below and refs. 22 and 24).…”

Section: Resultsmentioning

confidence: 99%

“…In this study we used MS for the quantification of PI3K-dependent protein kinase activity toward a substrate that is highly selective for this pathway, thereby directly quantifying a biochemical property of protein kinases that has not been investigated by MS thus far to a significant extent. We have found that this approach allows for the specific and sensitive quantification of pathway activation in absolute units, an important requirement for clinical analysis and a limitation of other detection methods, such as those based on radioactive and fluorescence techniques (22,23).…”

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confidence: 99%

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Ultrasensitive and absolute quantification of the phosphoinositide 3-kinase/Akt signal transduction pathway by mass spectrometry

Cutillas

Khwaja

Graupera

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The phosphoinositide 3-kinase (PI3K)͞Akt pathway controls a vast array of normal physiological processes and is frequently aberrantly activated in cancer, thus identifying PI3K͞Akt-signaling components as promising drug targets in oncology. However, implementation of rational cancer therapies for this pathway needs robust and simple tools to stratify patients according to PI3K pathway activation and to validate and measure the impact of targeted inhibition on primary cancer tissues. Herein we present a technique for the quantification of the PI3K͞Akt-signaling pathway based on the mass spectrometric measurement of PI3K-dependent protein kinase activity in cell lysates. The concept of this application of MS is to exploit enzymatic activity to amplify the signal of the enzyme under study analogous to the PCR used to amplify nucleic acid sequences. We show that this approach allows quantitative analysis of a cell-signaling pathway with high sensitivity, precision of quantification, and specificity. Due to its special analytical capabilities and potential for multiplexing, this approach could contribute significantly to cell-signaling studies and to the development and implementation of personalized cancer therapies.quantitative analysis ͉ signaling pathway ͉ personalized therapies ͉ cancer stem cells ͉ patient stratification S ignal transduction pathways downstream of receptor tyrosine kinases control a large array of biological processes, including energy metabolism, cell proliferation, survival, cell motility, and immune responses. One of these pathways, the phosphoinositide 3-kinase (PI3K)͞Akt axis, is one of the most frequently deregulated pathways in cancer (1-4) by virtue of oncogenic mutation, by overexpression of its signaling components (5-8), by inactivation of the PTEN tumor-suppressor protein, which opposes PI3K action (9-12), or by being at the crossroads of upstream oncogenic signals (reviewed in ref. 4).Because of the importance of this pathway in the control of several biological and pathological processes (13-15), robust methods to quantify its activation are needed. PI3K catalyzes the phosphorylation of the lipid phosphatidylinositol-4,5-bisphosphate to form phosphatidylinositol-3,4,5-trisphosphate, a second messenger that is needed for the activation of downstream Ser͞Thr kinase protein kinases, such as Akt [also known as protein kinase B (PKB)] and phosphoinositide-dependent kinase-1. The assessment of lipid production by various approaches has turned out to be cumbersome, and it is thus far not possible to directly measure production of phosphatidylinositol-3,4,5-trisphosphate in tissue samples. Activation of the PI3K pathway is therefore commonly assessed by examination of signaling components downstream of PI3K. These components include Akt͞PKB and kinases farther downstream of the Akt͞PKB cascade, such as p70-S6K. Most often, this examination is performed by indirect assessment using immunochemical methods that measure the phosphorylation status of Akt on Ser-473 or Thr-308 or of S6, a ribos...

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Ultrasensitive and absolute quantification of the phosphoinositide 3-kinase/Akt signal transduction pathway by mass spectrometry

Cutillas

Khwaja

Graupera

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…AKT activity assays were done as described previously (17) with three different peptide substrates: RPRAATF (17), Crosstide (GRPRTSSFAEG; ref. 18), and RIRTQFSFSLQERQLRG (16).…”

Section: Methodsmentioning

confidence: 99%

A Specific Role for AKT3 in the Genesis of Ovarian Cancer through Modulation of G2-M Phase Transition

et al. 2006

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Ovarian cancer is the major cause of death from gynecological malignancy, and there is an urgent need for new therapeutic targets. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been strongly implicated in the genesis of ovarian cancer. However, to identify and evaluate potential targets for therapeutic intervention, it is critical to understand the mechanism by which the PI3K/AKT pathway facilitates ovarian carcinogenesis. Here, we show that AKT3 is highly expressed in 19 of 92 primary ovarian tumors. Strikingly, purified AKT3 exhibited up to 10-fold higher specific activity than AKT1, potentially amplifying the effects of AKT3 overexpression. Consistent with this finding, AKT3 levels in a range of ovarian cancer cell lines correlated with total AKT activity and proliferation rates, implicating AKT3 as a key mediator of ovarian oncogenesis. Specific silencing of AKT3 using short hairpin RNA markedly inhibited proliferation of the two cell lines with highest AKT3 expression and total AKT activity, OVCA429 and DOV13, by slowing G 2 -M phase transition. These findings are consistent with AKT3 playing a key role in the genesis of at least one subset of ovarian cancers. (Cancer Res 2006; 66(24): 11718-25)

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“…However, these substrates are phosphorylated by other kinases such as p70 s6k and the mitogen-activated protein kinase (MAPK) kinase-activated protein kinases 1, and are not specific for Akt. Recently, Bozinovski et al [24] reported the use of the RPRAATF substrate peptide for the specific assay of the Akt activity in cell extracts.…”

Section: Analysis Of Pc12 Cells For Akt Activation Levelsmentioning

confidence: 99%

Method development and measurements of endogenous serine/threonine Akt phosphorylation using capillary electrophoresis for systems biology

2005

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Signal transduction studies have indicated that Akt is essential for transducing the signals originating from extracellular stimuli. An exploration of the Akt signal transduction mechanism depends on the ability to assay its activation states by determining the ability of Akt to phosphorylate various substrates. This paper describes a CE-based kinase assay for Akt using a UV detection method. The RPRAATF peptide was used as the specific substrate to determine the Akt activity. Under the CE separation conditions used, the phosphorylated and nonphosphorylated forms of the RPRAATF peptide were rapidly resolved in the Akt reaction mixture within 20 min. Using this method for measuring the Akt activity, the incubation time for the Akt reactions as well as the kinetic parameters (KM) were examined. Furthermore, the developed method was applied to a PC12 cell system to assess the dynamics of the Akt activity by examining the effectiveness of the RPRAATF peptide substrate under various cytokine-stimulated environments. These results highlight the feasibility of the CE method, which is a simple and reliable technique for determining and characterizing various enzyme reactions particularly kinase enzymes.

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The Synthetic Peptide RPRAATF Allows Specific Assay of Akt Activity in Cell Lysates

Cited by 17 publications

References 29 publications

Ultrasensitive and absolute quantification of the phosphoinositide 3-kinase/Akt signal transduction pathway by mass spectrometry

Ultrasensitive and absolute quantification of the phosphoinositide 3-kinase/Akt signal transduction pathway by mass spectrometry

A Specific Role for AKT3 in the Genesis of Ovarian Cancer through Modulation of G2-M Phase Transition

Method development and measurements of endogenous serine/threonine Akt phosphorylation using capillary electrophoresis for systems biology

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