Abstract
Phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are lipid second messengers that regulate various cellular processes by recruiting a wide range of downstream effector proteins to membranes. Several pleckstrin homology (PH) domains have been reported to interact with PtdIns(3,4)P2 and PtdIns(3,4,5)P3. To understand how these PH domains differentially respond to PtdIns(3,4)P2 and PtdIns(3,4,5)P3 signals, we quantitatively determined the PtdIns(3,4)P2 and PtdIns(3,4,5)P3 binding properties of several PH domains, including Akt, ARNO, Btk, DAPP1, Grp1, and C-terminal TAPP1 PH domains by surface plasmon resonance and monolayer penetration analyses. The measurements revealed that these PH domains have significant different phosphoinositide specificities and affinities. Btk-PH and TAPP1-PH showed genuine PtdIns(3,4,5)P3 and PtdIns(3,4)P2 specificities, respectively, whereas other PH domains exhibited less pronounced specificities. Also, the PH domains showed different degrees of membrane penetration, which greatly affected the kinetics of their membrane dissociation. Mutational studies showed that the presence of two proximal hydrophobic residues on the membrane-binding surface of the PH domain is important for membrane penetration and sustained membrane residence. When NIH 3T3 cells were stimulated with platelet-derived growth factor to generate PtdIns(3,4,5)P3, reversible translocation of Btk-PH, Grp1-PH, ARNO-PH, DAPP1-PH, and its L177A mutant to the plasma membrane was consistent with their in vitro membrane binding properties. Collectively, these studies provide new insight into how various PH domains would differentially respond to cellular PtdIns(3,4)P2 and PtdIns(3,4,5)P3 signals.
Highlights
PIs are minor components of membrane lipids, they regulate a wide range of biological processes, including cell proliferation, cell survival, differentiation, signal transduction, cytoskeleton organization, and membrane trafficking (1–3)
Unlike PtdIns(3,4,5)P3 that is primarily present in the plasma membrane, PtdIns(3,4)P2 may be localized at endosomes, intralumiphospholipase C; PX, phox homology; PH, pleckstrin homology domain; SPR, surface plasmon resonance; PDGF, platelet-derived growth factor; CFP, cyan fluorescence protein; DMEM, Dulbecco’s modified Eagle’s medium; EGFP, enhanced fluorescence green protein; MES, 4-morpholineethanesulfonic acid; Btk, Bruton tyrosine kinase
PH domains show low amino acid sequence homology, they have a conserved structural fold that consists of a 7-stranded -sandwich (1 to 7), one end of which is capped by a C-terminal ␣-helix, and the open end is connected by three variable loops (1–2, 3–4, and 6 –7 loops)
Summary
Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-snglycero-3-phosphoserine were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Protein Data Bank codes for the structures shown here are as follows: 1MAI (PLC␦) (68), 1FAQ (DAPP1) (66), 1EAZ (TAPP1c) (67), 1B55 (Btk) (72), 1U27 (ARNO) (70), 1FGY (Grp1) (71), and 1H10 (Akt) (69). For the expression of the Grp[1], Btk, ARNO, and DAPP1 PH domains, 1 liter of Luria broth containing 100 g/ml ampicillin was inoculated with BL21(DE3) cells harboring each construct, and the culture was grown at 37 °C until absorbance at 600 nm reached 0.5. At this time, 1 mM of isopropyl 1-thio--D-galactopyranoside was added, and cells were incubated at 25 °C for 14 h. Grp1-PH Akt-PH ARNO-PH Btk-PH TAPP1c-PH wild type TAPP1c-PH R211A DAPP1-PH wild type DAPP1-PH L177A DAPP1-PH V178A DAPP1-PH R184A a Not measurable
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