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Chemical genetics applied to the search for kinase protein substrates :A new class of triphosphate analogs.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Chemical genetics applied to the search for kinase protein substrates :
Reminder of title:	A new class of triphosphate analogs.
Author:	Kraybill, Brian C.
Description:	135 p.
Notes:	Adviser: Kevan M. Shokat.
Notes:	Source: Dissertation Abstracts International, Volume: 64-01, Section: B, page: 0214.
Contained By:	Dissertation Abstracts International64-01B.
Subject:	Chemistry, Organic.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3078633
ISBN:	0493995102

Chemical genetics applied to the search for kinase protein substrates :A new class of triphosphate analogs.
Kraybill, Brian C.

Chemical genetics applied to the search for kinase protein substrates :A new class of triphosphate analogs. [electronic resource] - 135 p.

Adviser: Kevan M. Shokat.

Thesis (Ph.D.)--Princeton University, 2003.

A third attempt yielded a new class of orthogonal triphosphate substrate analogs for the direct labeling of analog specific kinase protein targets. These analogs were constructed as derivatives of the Src family kinase inhibitor PP1 and were designed based on the crystal structures of PP1 bound to HCK and N6 (benzyl) ADP bound to c-Src (T338G). 3-Benzylpyrazolopyrimidine triphosphate (3-benzyl-PPTP) proved to be a substrate for a mutant of the MAP kinase p38 (p38-T106G/A157L/L167A). 3-Benzyl-PPTP was also shown to be preferred by v-Src (T338G) (kcat/KM = 3.2 x 10 6 min-1M-1) over ATP or the previously described ATP analog, N6 (benzyl) ATP. For the kinase CDK2 (F80G)/cyclin E, 3-benzyl-PPTP demonstrated catalytic efficiency (kcat/KM = 2.6 x 104 min -1M-1) comparable to ATP (kcat/K M = 5.0 x 104 min-1M -1) largely due to a significantly better KM (6.4 muM vs. 530 muM). In kinase protein substrate labeling experiments both 3-benzyl-PPTP and 3-phenyl-PPTP prove to be over 4 times more orthogonal than N6 (benzyl) ATP with respect to the wild type kinases found in murine spleenocyte cell lysates. These experiments also demonstrate that [gamma- 32P] 3-benzyl-PPTP is an excellent phosphodonor for labeling the direct protein substrates of CDK2 (F80G)/E in murine spleenocyte cell lysates, while competing with cellular levels (4 mM) of unlabelled ATP. This highly orthogonal nucleotide is accepted by three widely divergent kinases suggesting that it is likely to be generalizable across the entire kinase superfamily.

ISBN: 0493995102Subjects--Topical Terms:

193634
Chemistry, Organic.

Chemical genetics applied to the search for kinase protein substrates :A new class of triphosphate analogs.
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245 1 0 $a Chemical genetics applied to the search for kinase protein substrates : $b A new class of triphosphate analogs. $h [electronic resource]
300 $a 135 p.
500 $a Adviser: Kevan M. Shokat.
500 $a Source: Dissertation Abstracts International, Volume: 64-01, Section: B, page: 0214.
502 $a Thesis (Ph.D.)--Princeton University, 2003.
520 # $a A third attempt yielded a new class of orthogonal triphosphate substrate analogs for the direct labeling of analog specific kinase protein targets. These analogs were constructed as derivatives of the Src family kinase inhibitor PP1 and were designed based on the crystal structures of PP1 bound to HCK and N6 (benzyl) ADP bound to c-Src (T338G). 3-Benzylpyrazolopyrimidine triphosphate (3-benzyl-PPTP) proved to be a substrate for a mutant of the MAP kinase p38 (p38-T106G/A157L/L167A). 3-Benzyl-PPTP was also shown to be preferred by v-Src (T338G) (kcat/KM = 3.2 x 10 6 min-1M-1) over ATP or the previously described ATP analog, N6 (benzyl) ATP. For the kinase CDK2 (F80G)/cyclin E, 3-benzyl-PPTP demonstrated catalytic efficiency (kcat/KM = 2.6 x 104 min -1M-1) comparable to ATP (kcat/K M = 5.0 x 104 min-1M -1) largely due to a significantly better KM (6.4 muM vs. 530 muM). In kinase protein substrate labeling experiments both 3-benzyl-PPTP and 3-phenyl-PPTP prove to be over 4 times more orthogonal than N6 (benzyl) ATP with respect to the wild type kinases found in murine spleenocyte cell lysates. These experiments also demonstrate that [gamma- 32P] 3-benzyl-PPTP is an excellent phosphodonor for labeling the direct protein substrates of CDK2 (F80G)/E in murine spleenocyte cell lysates, while competing with cellular levels (4 mM) of unlabelled ATP. This highly orthogonal nucleotide is accepted by three widely divergent kinases suggesting that it is likely to be generalizable across the entire kinase superfamily.
520 # $a An N7 (methyl) adenine analog was not stable to triphosphate synthesis. Diazepine nucleotides were shown to bind to v-Src mutants, but were not catalytically viable. These first two attempts failed to produce viable ATP analog substrates.
520 # $a Elucidation of protein kinase signaling networks is challenging due to the large size of the protein kinase superfamily (>500 human kinases). We answer this challenge with a novel chemical gentic approach. First, adenosine triphosphate (ATP) analogs that are not substrates for any kinases are developed. Second, a selected kinase is mutated to accept the ATP analog(s) (called an analog specific kinase). Finally, the [gamma-32P] ATP analog is used in conjunction with the analog specific kinase to label the direct protein substrates.
590 $a School code: 0181.
650 # 0 $a Chemistry, Organic. $3 193634
650 # 0 $a Chemistry, Biochemistry. $3 226900
650 # 0 $a Biology, Molecular. $3 226919
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773 0 # $g 64-01B. $t Dissertation Abstracts International
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790 1 0 $a Shokat, Kevan M., $e advisor
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