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. 2009 Feb 9;184(3):451-62.
doi: 10.1083/jcb.200807047.

Phospholipase C-mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane

Jian-Jiang Hao  1 , Yin Liu, Michael Kruhlak, Karen E Debell, Barbara L Rellahan, Stephen Shaw
Affiliations

Phospholipase C-mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane

Jian-Jiang Hao et al. J Cell Biol. .

Abstract

Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1-mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation.

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Figures

Figure 1.
Figure 1.
SDF-1–induced ERM protein dephosphorylation depends on PLC signaling. (A) Detection of ERM protein phosphorylation by WB in SDF-1–stimulated PBT. Freshly isolated PBTs from a healthy donor were preincubated with PLC inhibitor U73122, its nonfunctional analogue U73433, or PI3-K inhibitor Ly294002 for 10 min and stimulated with/without SDF-1 or PLC activator m-3M3FBS for 45 s. Note that phosphorylated moesin and ezrin are both present in PBT but run as a single band under these conditions. (B) Detection of both moesin and ezrin protein dephosphorylation by WB in SDF-1–stimulated PBT. The samples were prepared as in A but were resolved by running on SDS 4–20% Tris-glycine gels to separate moesin from ezrin. The top panel shows a blot for pERM; the top band is ezrin, and the bottom band is moesin. The middle and bottom panels show WB with antiezrin rabbit polyclonal antibody and antiezrin mouse mAb. (C) Quantitative analysis of data from B using Odyssey software (version 3.0; LI-COR Biosciences). CT, control.
Figure 2.
Figure 2.
SDF-1–induced delocalization of ERM proteins from plasma membrane depends on PLC signaling. Immunofluorescence analysis of PBT treated as in Fig. 1. After treatments, the cells were subjected to fixation and immunofluorescence staining. The third row shows an overlay of CD44 and pERM, and the fourth row shows a higher magnification of boxed regions in the third row, which highlight either colocalization of pERM with CD44 or lack thereof. The fifth row shows differential interference contrast (DIC) images displaying cell polarization. CT, control.
Figure 3.
Figure 3.
SDF-1–induced ERM protein release from the membrane-enriched fraction depends on PLC signaling. (A) Freshly isolated PBTs were pretreated with the indicated inhibitors at 37°C for 15 min, and cells were stimulated with SDF-1 at 37°C for 45 s. Subcellular fractionation by sonication and centrifugation was performed as described in Materials and methods. Total indicates postnuclear supernatant, S100 indicates soluble cytosolic fraction (S100) thereof obtained by centrifugation of postnuclear supernatant at 100,000 g, and P100 indicates pellet (P100) whose enrichment in membrane is documented by WB for MHC-I. White line indicates that intervening lanes have been spliced out. (B) Quantitative analysis of data from A by Odyssey software (version 3.0). SDF-1 stimulation induces release of both moesin and ezrin from membrane. This release can only be prevented by PLC inhibitor U73122 but not by PI3-K inhibitor. CT, control.
Figure 4.
Figure 4.
Transfection with constitutively active PLC induces ERM protein dephosphorylation and release from the plasma membrane. (A) Jurkat cells were transfected with the indicated PLC-γ1 constructs (NN, constitutively active; or Y783F, inactive) or construct-encoding GFP-PH and analyzed by WB. Expression of PLC-γ1 in Jurkat does not influence the expression of moesin constructs (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200807047/DC1). (B) Imaging of control cells (top) compared with cells transfected with constitutively active NN PLC-γ1 construct (middle) or with inactive PLC-γ1 Y783F (bottom). Markers examined are the transfected PLC (anti-HA antibody, purple), pERM level (red), and transfected GFP-PH localization. (C) Fluorescent analysis of Jurkat cells cotransfected with GFP-moesin (wt or T558D) and PLC-γ1 constructs (detected with anti-HA antibody [red]). (D) Quantitative analysis of data from C (n = 10 for each condition). Quantitative analysis was performed as described in Materials and methods. (E) SDF-1 stimulation induces hydrolysis of PIP2. Jurkat cells were transfected with GFP-tagged PH domain construct and analyzed by immunofluorescence microscopy after SDF-1 or PLC activator treatment. Note that unlike PBTs, Jurkat cells do not undergo striking shape change (polarization) in response to SDF-1. Error bars indicate SEM. DIC, differential interference contrast.
Figure 5.
Figure 5.
Membrane translocation of type IV 5-ptase reduces plasma membrane PIP2 and releases moesin and ezrin from the membrane. (A) Jurkat cells were transfected with GFP-tagged PH domain or the indicated GFP-moesin or GFP-ezrin constructs, along with the membrane-targeted FRB-CFP and the mRFP-FKBP domain constructs (with or without type IV 5-ptase domain as indicated), and were analyzed by fluorescence microscopy before and after rapamycin (Rapa) treatment. GFP fluorescence is shown. mRFP-FKBP, mRFP-FKBP– 5-ptase, and FRB-CFP fluorescence images are shown in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200807047/DC1). (B) Quantitative analysis of the membrane enrichment of moesin, ezrin, or PH domain constructs (n = 10 for each condition) was performed as described in Materials and methods. Error bars indicate SEM.
Figure 6.
Figure 6.
PIP2 binding is required for the association of moesin with the cytoplasmic tails of CD44, CD43, ICAM2, and ICAM3. (A) Effects of phospholipids or IP3 on the interaction between moesin and cytoplasmic tails of CD44, CD43, ICAM2, and ICAM3. The bound proteins by GST-tagged tails in the pellet were detected with antimoesin antibody and are shown in the top panel. Arrowheads indicate the intact protein in the lanes where degredation products also exist. (B) The effects of T558D mutation mimicking phosphorylation, K4N mutations, or their combination on the activation of moesin for CD44 tail binding. The bound proteins shown in the top panel were detected by WB. Further characterization of the recombinant protein is shown in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200807047/DC1). GST (as control) or GST fusion proteins in the pellet were detected with Coomassie blue staining and are shown in the bottom panels. Each lane represents 40% of the amount of protein in the pellet. PI, phosphatidylinositol; PS, phosphatidylserine.
Figure 7.
Figure 7.
Type IV 5-ptase mediates decrease of PIP2 and induces endogenous ERM protein dephosphorylation in Jurkat cells. (A) Immunofluorescence analysis of ERM protein phosphorylation in cells transfected with PH-GFP construct, the membrane-targeted FRB-CFP, and the mRFP-FKBP domain construct (with or without type IV 5-ptase domain as indicated). (B) Quantitative analysis of pERM (n = 10 for each condition). Error bars indicate SEM. Rapa, rapamycin; CT, control.
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