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Structural basis for the function of the Bcr-Abl protein substrate CrkL and human-CrkII as a novel partner for Cyclophilin A
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Title
Structural basis for the function of the Bcr-Abl protein substrate CrkL and human-CrkII as a novel partner for Cyclophilin A
Name
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Jankowski
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Wojciech
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1979-
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Wojciech Jankowski
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author
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Kalodimos
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Charalampos G
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Charalampos G Kalodimos
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Advisory Committee
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chair
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David A
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David A Case
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Advisory Committee
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internal member
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Olson
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Wilma K
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Wilma K Olson
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Advisory Committee
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internal member
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Birge
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Raymond
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Raymond Birge
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Advisory Committee
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outside member
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Rutgers University
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degree grantor
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Graduate School - New Brunswick
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school
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Text
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theses
OriginInfo
DateCreated
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2012
DateOther
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2012-10
Place
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xx
Language
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eng
Subject
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Topic
Animal Sciences
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TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
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ETD_4170
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electronic resource
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application/pdf
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text/xml
Extent
xxix, 154 p. : ill.
Note
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Ph.D.
Note
(type = bibliography)
Includes bibliographical references
Note
(type = vita)
Includes vita
Note
(type = statement of responsibility)
by Wojciech Jankowski
Subject
(authority = ETD-LCSH)
Topic
Protein binding
Subject
(authority = ETD-LCSH)
Topic
Nuclear magnetic resonance spectroscopy
Identifier
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http://hdl.rutgers.edu/1782.1/rucore10001600001.ETD.000066832
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Title
Graduate School - New Brunswick Electronic Theses and Dissertations
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rucore19991600001
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(displayLabel = Rutgers, The State University of New Jersey)
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Identifier
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doi:10.7282/T3668BZR
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(authority = ExL-Esploro)
ETD doctoral
Abstract
Adaptor proteins are known to play an essential role in assembling protein-protein complexes which result in cellular signal propagation. Crk belongs to a family of adaptor proteins and was originally identified as an oncogene product of the CT10 retrovirus (v-Crk). Cellular homologues of v-Crk include CrkI, CrkII and CrkL. CrkI and CrkII are different splice variants, whereas CrkL is encoded by a distinct gene. Crk proteins contain one Src homology 2 (SH2) and one or two Src homology 3 (SH3) domains. Specific domain organization can assemble and activate a number of different ligands, including Abl. It remains poorly understood why CrkII and CrkL have distinct physiological roles despite showing similar domain structures, high sequence identity, and identical binding partners. Unlike CrkII, CrkL was found to be a key signaling molecule to interact with Bcr-Abl, which is a tyrosine kinase that plays a major role in Chronic Myeloid Leukemia(CML) pathogenesis. The interaction of CrkL with Bcr-Abl and its potent tyrosine phosphorylation (higher as compared to CrkII) is commonly used as a hallmark of Bcr-Abl kinase activity and response to tyrosine kinase inhibitors used in CML treatment. Knowing the differences in 3-dimensional structures between CrkII and CrkL would help to understand how these adaptors alter key signaling partners. However, the structure of CrkL is not available.
Using NMR spectroscopy methodologies complemented by many other biochemical and biophysical techniques, we show that CrkL and phosphorylated CrkL structures are radically different from the corresponding structures of CrkII. The phosphorylation of Tyr221 (CrkII) and Tyr207 (CrkL) by Abl induces intramolecular binding to the SH2 domain, which in the case of phosphorylated CrkII was shown to completely abrogate signal transduction. In phosphorylated CrkL, however, the SH3N domain remains accessible and can form complexes. The data show that CrkL, unlike CrkII, forms a constitutive complex with Abl hence explaining the preference of Bcr-Abl for CrkL over CrkII. The results also highlight how the structural organization of the modular domains in adaptor proteins can control signaling outcome.
In the second part we show that the Gly219−Pro220 motif of CrkII binds into theactive site cleft of CypA. In contrast, CrkL does not contain this GP motif and therefore, is not susceptible to CypA regulation. The interaction between CypA and CrkII occurs both in vitro and in vivo. CypA is recruited to the CrkII phosphorylation site (Tyr221), and delays phosphorylation by Abl. This is a novel role for CypA, which appears to act as a selective switch to modulate the level of phosphorylation of a signaling protein.
Using NMR spectroscopy methodologies complemented by many other biochemical and biophysical techniques, we show that CrkL and phosphorylated CrkL structures are radically different from the corresponding structures of CrkII. The phosphorylation of Tyr221 (CrkII) and Tyr207 (CrkL) by Abl induces intramolecular binding to the SH2 domain, which in the case of phosphorylated CrkII was shown to completely abrogate signal transduction. In phosphorylated CrkL, however, the SH3N domain remains accessible and can form complexes. The data show that CrkL, unlike CrkII, forms a constitutive complex with Abl hence explaining the preference of Bcr-Abl for CrkL over CrkII. The results also highlight how the structural organization of the modular domains in adaptor proteins can control signaling outcome.
In the second part we show that the Gly219−Pro220 motif of CrkII binds into theactive site cleft of CypA. In contrast, CrkL does not contain this GP motif and therefore, is not susceptible to CypA regulation. The interaction between CypA and CrkII occurs both in vitro and in vivo. CypA is recruited to the CrkII phosphorylation site (Tyr221), and delays phosphorylation by Abl. This is a novel role for CypA, which appears to act as a selective switch to modulate the level of phosphorylation of a signaling protein.
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The author owns the copyright to this work.
RightsHolder
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Name
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Jankowski
GivenName
Wojciech
Role
Copyright Holder
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Permission or license
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2012-07-08 21:45:42
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Name
Wojciech Jankowski
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Affiliation
Rutgers University. Graduate School - New Brunswick
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Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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DateTime
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2012-10-31
DateTime
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2014-10-31
Type
Embargo
Detail
Access to this PDF has been restricted at the author's request. It will be publicly available after October 31st, 2014.
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Copyright protected
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Open
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