Biochem. J. (2000) 351
(19–31) (Printed in Great Britain)
Research Communication
Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities
Simon DOWLER*, Richard A. CURRIE†, David G. CAMPBELL*, Maria DEAK‡, Gursant KULAR†, C. Peter DOWNES† and Dario R. ALESSI*1
*MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., †Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., and ‡Division of Signal Transduction Therapy, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P3. The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P3. Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P3, but instead possess unexpected and novel phosphoinositide-binding specificities in vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P2 [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P2 (centaurin-b2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s).
Key words: AtPH1, centaurin-b2, phosphoinositide 3-kinase, PtsIns3P-binding PH domain protein-1 (PEPP1), PtsIns4P adaptor protein-1 (FAPP1), tandem pleckstrin-homology (PH)-domain-containing protein-1 (TAPP1), TAPP2.
Abbreviations used: PI 3-kinase, phosphoinositide 3-kinase; PH, pleckstrin homology; PKB, protein kinase B; PDK1, 3-phosphoinositide-dependent protein kinase-1; BTK, Bruton's tyrosine kinase; DAPP1, dual adaptor for phosphotyrosine and 3-phosphoinositides; ARF, ADP-ribosylation factor; GAP, GTPase-activating protein; PPBM, putative phosphatidylinositol 3,4,5-trisphosphate-binding motif; EST, expressed sequence tag; GST, glutathione S-transferase; TAPP, tandem PH-domain-containing protein; FAPP1, phosphatidylinositol-four-phosphate adaptor protein-1; PEPP1, phosphatidylinositol-three-phosphate-binding PH-domain protein-1; HEK-293, human embryonic kidney-293; NCBI, National Center for Biotechnology Information; PDZ, postsynaptic density protein (PSD-95)/Drosophila disc large-tumour suppressor (Dlg)/tight junction protein (ZO1).
1To whom correspondence should be addressed (e-mail s.j.dowler@dundee.ac.uk).
The nucleotide sequence data reported will appear in the National Center for Biotechnology Information database under the accession nos. as shown: human TAPP1 (full-length sequence), AF286160; human TAPP2 (partial sequence), AF286164; mouse TAPP2 (full-length sequence), AF286161; human FAPP1 (full-length sequence), AF286162; mouse FAPP1 (full-length sequence), AF286163; human PEPP1 (full-length sequence), AY007233.
Received 17 July 2000/16 August 2000; accepted 22 August 2000
The Biochemical Society, London ©
2000
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