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Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain.
J Biol Chem. 2003 Oct 10; 278(41):39489-96.JB

Abstract

Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1m and GAP1IP4BP are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1m PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas the domain from GAP1IP4BP binds PtdIns(3,4,5)P3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1m is cytosolic and undergoes a rapid PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1IP4BP is constitutively associated, in a PtdIns(4,5)P2-dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261-28268). In the present study, we have used molecular modeling to identify residues in the GAP1IP4BP PH domain predicted to be required for high affinity binding to PtdIns(4,5)P2. This has allowed the isolation of a mutant, GAP1IP4BP-(K591T), which while retaining high affinity for PtdIns(3,4,5)P3 has a 6-fold reduction in its affinity for PtdIns(4,5)P2. Importantly, GAP1IP4BP-(K591T) is predominantly localized to the cytosol and undergoes a PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1IP4BP PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1IP4BP.

Authors+Show Affiliations

Inositide Group, Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12885767

Citation

Cozier, Gyles E., et al. "Engineering the Phosphoinositide-binding Profile of a Class I Pleckstrin Homology Domain." The Journal of Biological Chemistry, vol. 278, no. 41, 2003, pp. 39489-96.
Cozier GE, Bouyoucef D, Cullen PJ. Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain. J Biol Chem. 2003;278(41):39489-96.
Cozier, G. E., Bouyoucef, D., & Cullen, P. J. (2003). Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain. The Journal of Biological Chemistry, 278(41), 39489-96.
Cozier GE, Bouyoucef D, Cullen PJ. Engineering the Phosphoinositide-binding Profile of a Class I Pleckstrin Homology Domain. J Biol Chem. 2003 Oct 10;278(41):39489-96. PubMed PMID: 12885767.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain. AU - Cozier,Gyles E, AU - Bouyoucef,Dalila, AU - Cullen,Peter J, Y1 - 2003/07/28/ PY - 2003/7/30/pubmed PY - 2003/12/3/medline PY - 2003/7/30/entrez SP - 39489 EP - 96 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 278 IS - 41 N2 - Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1m and GAP1IP4BP are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1m PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas the domain from GAP1IP4BP binds PtdIns(3,4,5)P3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1m is cytosolic and undergoes a rapid PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1IP4BP is constitutively associated, in a PtdIns(4,5)P2-dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261-28268). In the present study, we have used molecular modeling to identify residues in the GAP1IP4BP PH domain predicted to be required for high affinity binding to PtdIns(4,5)P2. This has allowed the isolation of a mutant, GAP1IP4BP-(K591T), which while retaining high affinity for PtdIns(3,4,5)P3 has a 6-fold reduction in its affinity for PtdIns(4,5)P2. Importantly, GAP1IP4BP-(K591T) is predominantly localized to the cytosol and undergoes a PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1IP4BP PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1IP4BP. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/12885767/Engineering_the_phosphoinositide_binding_profile_of_a_class_I_pleckstrin_homology_domain_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=12885767 DB - PRIME DP - Unbound Medicine ER -