Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays
The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and...
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| Veröffentlicht in: | Analytical biochemistry 2011-07, Vol.414 (2), p.179-186 |
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| creator | Hofler, Alexandra Nichols, Tim Grant, Stephan Lingardo, Laura Esposito, Edward A. Gridley, Scott Murphy, Sean T. Kath, John C. Cronin, Ciarán N. Kraus, Michelle Alton, Gordon Xie, Zhi Sutton, Scott Gehring, Mike Ermolieff, Jacques |
| description | The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP
3) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1. |
| doi_str_mv | 10.1016/j.ab.2011.03.013 |
| format | Article |
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3) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/j.ab.2011.03.013</identifier><identifier>PMID: 21402045</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>3-Phosphoinositide-Dependent Protein Kinases ; Adenine - analogs & derivatives ; Adenine - chemistry ; Adenine - pharmacology ; AKT ; Animals ; carcinogenesis ; cell growth ; cells ; CHO Cells ; Cricetinae ; Cricetulus ; Enzyme Assays - methods ; Histidine - genetics ; Histidine - metabolism ; Humans ; Kinase inhibitor ; Kinetics ; membranes ; Oligopeptides - genetics ; Oligopeptides - metabolism ; PDK1 ; Phosphorylation ; plasma membrane ; plasma membranes ; Protein Kinase Inhibitors - chemistry ; Protein Kinase Inhibitors - pharmacology ; Protein-Serine-Threonine Kinases - antagonists & inhibitors ; Protein-Serine-Threonine Kinases - genetics ; Protein-Serine-Threonine Kinases - metabolism ; Proto-Oncogene Proteins c-akt - antagonists & inhibitors ; Proto-Oncogene Proteins c-akt - genetics ; Proto-Oncogene Proteins c-akt - metabolism ; Pyrazines - chemistry ; Pyrazines - pharmacology ; Recombinant Proteins - antagonists & inhibitors ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Signal Transduction ; tagging ; Template-directed assembly (TDA)</subject><ispartof>Analytical biochemistry, 2011-07, Vol.414 (2), p.179-186</ispartof><rights>2011 Elsevier Inc.</rights><rights>Copyright © 2011 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c439t-a5cbb1a2f8163dfe51eccd9613bb8aa5aae960b0c12f1dc7eda8c15eba50f27c3</citedby><cites>FETCH-LOGICAL-c439t-a5cbb1a2f8163dfe51eccd9613bb8aa5aae960b0c12f1dc7eda8c15eba50f27c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0003269711001709$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21402045$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hofler, Alexandra</creatorcontrib><creatorcontrib>Nichols, Tim</creatorcontrib><creatorcontrib>Grant, Stephan</creatorcontrib><creatorcontrib>Lingardo, Laura</creatorcontrib><creatorcontrib>Esposito, Edward A.</creatorcontrib><creatorcontrib>Gridley, Scott</creatorcontrib><creatorcontrib>Murphy, Sean T.</creatorcontrib><creatorcontrib>Kath, John C.</creatorcontrib><creatorcontrib>Cronin, Ciarán N.</creatorcontrib><creatorcontrib>Kraus, Michelle</creatorcontrib><creatorcontrib>Alton, Gordon</creatorcontrib><creatorcontrib>Xie, Zhi</creatorcontrib><creatorcontrib>Sutton, Scott</creatorcontrib><creatorcontrib>Gehring, Mike</creatorcontrib><creatorcontrib>Ermolieff, Jacques</creatorcontrib><title>Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP
3) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.</description><subject>3-Phosphoinositide-Dependent Protein Kinases</subject><subject>Adenine - analogs & derivatives</subject><subject>Adenine - chemistry</subject><subject>Adenine - pharmacology</subject><subject>AKT</subject><subject>Animals</subject><subject>carcinogenesis</subject><subject>cell growth</subject><subject>cells</subject><subject>CHO Cells</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>Enzyme Assays - methods</subject><subject>Histidine - genetics</subject><subject>Histidine - metabolism</subject><subject>Humans</subject><subject>Kinase inhibitor</subject><subject>Kinetics</subject><subject>membranes</subject><subject>Oligopeptides - genetics</subject><subject>Oligopeptides - metabolism</subject><subject>PDK1</subject><subject>Phosphorylation</subject><subject>plasma membrane</subject><subject>plasma membranes</subject><subject>Protein Kinase Inhibitors - chemistry</subject><subject>Protein Kinase Inhibitors - pharmacology</subject><subject>Protein-Serine-Threonine Kinases - antagonists & inhibitors</subject><subject>Protein-Serine-Threonine Kinases - genetics</subject><subject>Protein-Serine-Threonine Kinases - metabolism</subject><subject>Proto-Oncogene Proteins c-akt - antagonists & inhibitors</subject><subject>Proto-Oncogene Proteins c-akt - genetics</subject><subject>Proto-Oncogene Proteins c-akt - metabolism</subject><subject>Pyrazines - chemistry</subject><subject>Pyrazines - pharmacology</subject><subject>Recombinant Proteins - antagonists & inhibitors</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Signal Transduction</subject><subject>tagging</subject><subject>Template-directed assembly (TDA)</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU1P3DAQhq2qqGyh955a37iQMI7z5d7QtoUKJJAWztbYmWy8ysc2Tqj239erUG49jUZ63nekZxj7LCAWIPKrXYwmTkCIGGQMQr5jKwEqj0CCes9WACCjJFfFKfvo_Q4CmGb5B3aaiBQSSLMV6zbTXB34UPOpIf74_U5cXd89ce-2Pbau3_I9Ts0fPPDZHzdPLdnJvSwod33jjJuG0V_y2_XmkmNfceob7C1V3LjBNtQ5iy1H7_Hgz9lJja2nT6_zjD3__PG0vo3uH25-ra_vI5tKNUWYWWMEJnUpclnVlAmytlK5kMaUiBkiqRwMWJHUorIFVVhakZHBDOqksPKMXSy9-3H4PZOfdOe8pbbFnobZ6zIvCqEgLQIJC2nHwfuRar0fXYfjQQvQR8d6p9Hoo2MNUgfHIfLltXw2HVVvgX9SA_B1AWocNG5H5_XzJjTk4R-JUrIMxLeFoCDhxdGovXV0lObG4FdXg_v__b9hfZUV</recordid><startdate>20110715</startdate><enddate>20110715</enddate><creator>Hofler, Alexandra</creator><creator>Nichols, Tim</creator><creator>Grant, Stephan</creator><creator>Lingardo, Laura</creator><creator>Esposito, Edward A.</creator><creator>Gridley, Scott</creator><creator>Murphy, Sean T.</creator><creator>Kath, John C.</creator><creator>Cronin, Ciarán N.</creator><creator>Kraus, Michelle</creator><creator>Alton, Gordon</creator><creator>Xie, Zhi</creator><creator>Sutton, Scott</creator><creator>Gehring, Mike</creator><creator>Ermolieff, Jacques</creator><general>Elsevier Inc</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20110715</creationdate><title>Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays</title><author>Hofler, Alexandra ; Nichols, Tim ; Grant, Stephan ; Lingardo, Laura ; Esposito, Edward A. ; Gridley, Scott ; Murphy, Sean T. ; Kath, John C. ; Cronin, Ciarán N. ; Kraus, Michelle ; Alton, Gordon ; Xie, Zhi ; Sutton, Scott ; Gehring, Mike ; Ermolieff, Jacques</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c439t-a5cbb1a2f8163dfe51eccd9613bb8aa5aae960b0c12f1dc7eda8c15eba50f27c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>3-Phosphoinositide-Dependent Protein Kinases</topic><topic>Adenine - analogs & derivatives</topic><topic>Adenine - chemistry</topic><topic>Adenine - pharmacology</topic><topic>AKT</topic><topic>Animals</topic><topic>carcinogenesis</topic><topic>cell growth</topic><topic>cells</topic><topic>CHO Cells</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>Enzyme Assays - methods</topic><topic>Histidine - genetics</topic><topic>Histidine - metabolism</topic><topic>Humans</topic><topic>Kinase inhibitor</topic><topic>Kinetics</topic><topic>membranes</topic><topic>Oligopeptides - genetics</topic><topic>Oligopeptides - metabolism</topic><topic>PDK1</topic><topic>Phosphorylation</topic><topic>plasma membrane</topic><topic>plasma membranes</topic><topic>Protein Kinase Inhibitors - chemistry</topic><topic>Protein Kinase Inhibitors - pharmacology</topic><topic>Protein-Serine-Threonine Kinases - antagonists & inhibitors</topic><topic>Protein-Serine-Threonine Kinases - genetics</topic><topic>Protein-Serine-Threonine Kinases - metabolism</topic><topic>Proto-Oncogene Proteins c-akt - antagonists & inhibitors</topic><topic>Proto-Oncogene Proteins c-akt - genetics</topic><topic>Proto-Oncogene Proteins c-akt - metabolism</topic><topic>Pyrazines - chemistry</topic><topic>Pyrazines - pharmacology</topic><topic>Recombinant Proteins - antagonists & inhibitors</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Signal Transduction</topic><topic>tagging</topic><topic>Template-directed assembly (TDA)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hofler, Alexandra</creatorcontrib><creatorcontrib>Nichols, Tim</creatorcontrib><creatorcontrib>Grant, Stephan</creatorcontrib><creatorcontrib>Lingardo, Laura</creatorcontrib><creatorcontrib>Esposito, Edward A.</creatorcontrib><creatorcontrib>Gridley, Scott</creatorcontrib><creatorcontrib>Murphy, Sean T.</creatorcontrib><creatorcontrib>Kath, John C.</creatorcontrib><creatorcontrib>Cronin, Ciarán N.</creatorcontrib><creatorcontrib>Kraus, Michelle</creatorcontrib><creatorcontrib>Alton, Gordon</creatorcontrib><creatorcontrib>Xie, Zhi</creatorcontrib><creatorcontrib>Sutton, Scott</creatorcontrib><creatorcontrib>Gehring, Mike</creatorcontrib><creatorcontrib>Ermolieff, Jacques</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hofler, Alexandra</au><au>Nichols, Tim</au><au>Grant, Stephan</au><au>Lingardo, Laura</au><au>Esposito, Edward A.</au><au>Gridley, Scott</au><au>Murphy, Sean T.</au><au>Kath, John C.</au><au>Cronin, Ciarán N.</au><au>Kraus, Michelle</au><au>Alton, Gordon</au><au>Xie, Zhi</au><au>Sutton, Scott</au><au>Gehring, Mike</au><au>Ermolieff, Jacques</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>2011-07-15</date><risdate>2011</risdate><volume>414</volume><issue>2</issue><spage>179</spage><epage>186</epage><pages>179-186</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP
3) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>21402045</pmid><doi>10.1016/j.ab.2011.03.013</doi><tpages>8</tpages></addata></record> |
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| ispartof | Analytical biochemistry, 2011-07, Vol.414 (2), p.179-186 |
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| subjects | 3-Phosphoinositide-Dependent Protein Kinases Adenine - analogs & derivatives Adenine - chemistry Adenine - pharmacology AKT Animals carcinogenesis cell growth cells CHO Cells Cricetinae Cricetulus Enzyme Assays - methods Histidine - genetics Histidine - metabolism Humans Kinase inhibitor Kinetics membranes Oligopeptides - genetics Oligopeptides - metabolism PDK1 Phosphorylation plasma membrane plasma membranes Protein Kinase Inhibitors - chemistry Protein Kinase Inhibitors - pharmacology Protein-Serine-Threonine Kinases - antagonists & inhibitors Protein-Serine-Threonine Kinases - genetics Protein-Serine-Threonine Kinases - metabolism Proto-Oncogene Proteins c-akt - antagonists & inhibitors Proto-Oncogene Proteins c-akt - genetics Proto-Oncogene Proteins c-akt - metabolism Pyrazines - chemistry Pyrazines - pharmacology Recombinant Proteins - antagonists & inhibitors Recombinant Proteins - genetics Recombinant Proteins - metabolism Signal Transduction tagging Template-directed assembly (TDA) |
| title | Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays |
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