Localization,Translocation and Binding Properties of Murine PH Domains in RAW Cells T. Mukai, J. W. Whalen, W. Park, E. Gehrig, J. Zavzavadijian, I. Vadivelu, J. Zavzavadjian, E. Wall, A. Maer, G. Chandy and N. A. O’Rourke Alliance for Cell Signaling (AfCS) Microscopy Lab, Stanford University, AfCS Mol. Biology Lab, Caltech, AfCS, Bioinformatics Lab, UCSD Introduction The pleckstrin homology (PH) domain is a regulatory domain contained in many signaling proteins that binds to lipids and targets the proteins to the vicinity of their substrates or activators on cell membranes. The PH domain from Akt binds to PI(3,4,5)P 3 and PI(3,4)P 2.. Increases in the levels of PI(3,4,5)P 3 recruit the PH domain to the membrane, thus causing the full length protein to translocate from the cytosol to the plasma membrane. Tobias Meyer and colleagues generated a GFP-tagged version of the PH domain from Akt that serves as a “biosensor” for increases in PI(3,4,5)P 3 levels in the cell. To learn more about the role of PH domains in signaling proteins and develop novel “biosensors” for monitoring signaling events, we are attempting to clone all the PH domains present in mouse signaling proteins. So far, we cloned 135 PH domains and generated constructs with an N- terminal YFP or CFP tag for each one. For proteins with two PH domains, both of the PH domains are cloned separately and in some cases in tandem. The PH domain constructs were transfected into the RAW macrophage cell line. We screened the entire set of PH domain constructs in RAW cells to determine where they localize in quiescent cells. In addition,, we used time-lapse confocal microscopy in RAW cells to determine whether the PH domains translocate upon stimulation with 100 nM C5a. Finally, we used a lipid binding assay to determine the binding selectivity of each of the PH domains. The knowledge gained from these three complementary assays will add to our understanding of PH domains and aid in the design of new biosensors. Generation of PH Domain Constructs Included up to 50 AA of flanking sequence up to but not including neighboring domains. Each construct is N-terminally tagged with YFP and CFP. Two PH domains from 1 protein are denoted PH1A and PH2A. In six cases, tandem PH domains from a single protein were cloned with intervening sequence. PH Domain Localization in RAW Cells To determine the localization patterns of the PH domains, we transfected the constructs into the RAW cell line. The YFP- tagged proteins were visualized in the cells using a Nipkow lens-enhanced confocal microscope. We assayed the localization of 135 PH domain constructs in RAW cells. The majority, 72, localized to the cytosol and nucleus (Table, above top). The remainder were localized in the cytosol, nucleus and plasma membrane or in other combinations of organelles (Table, above bottom). These data are available on the AfCS website ( Localization of Multiple PH domains from One Protein In several instances, we screened the localization pattern of 2 PH domains from the same protein. The localization patterns for the PH domains from T-cell lymphoma invasion and metastasis 1 (Tiam-1) and Plekstrin (Plek) are shown above. For Tiam-1, the two PH domains are localized in the same pattern and the lipid bindig specificity of the two domains is similar. For Plekstrin, the first PH domain (PH1A) is localized to the plasma membrane, cytosol and nucleus, while the second PH domain (PH2A) is localized to the cytosol and nucleus only. In the lipid binding analysis (right), we found that Plek(PH1A) binds more specifically to PI(3,4)P 2 and PI(3,4,5)P 3 than PLEK(PH2A). PH domain Translocation in RAW Cells For our translocation studies, we transfected RAW cells with YFP-tagged PH domain constructs and then serum-deprived the cells 1-3 hours prior to performing the screen. The cells were placed on the confocal microscope stage and imaged every 10 seconds for 40 scans. After 5 scans, we stimulated the cells with C5a (100nM). We found that 10 PH domains translocated from the cytosol to the plasma membrane upon stimulation of the cells. Translocation of the Bam 32 and SH3BP2 PH domains RAW cells expressing either the YFP-tagged PH domains from the adaptor/scaffold proteins Bam32(above) or SH3BP2 (below) were stimulated with 100 nMC5a. The montage above shows 8 images from every 5 th scan (50 secs.) in a time series, with the addition of the ligand after the 50 seconds. The PH domains are found initially in the nucleus and cytosol. Upon cell stimulation, the PH domains translocate to the plasma membrane. Conclusions The majority of the PH domains are expressed throughout the cytosol and nucleus, while some are distributed in the plasma membrane or other organelles as well. We identified 10 PH domains that undergo translocation upon stimulation with 100 nM C5a. In the lipid binding study, we found that 29 of the PH domains show strong selective binding to a subset of the phosphoinosotides. Tiam-1 (PH2A) Tiam-1 (PH1A) Plek (PH1A)Plek (PH2A) Bam32 (PH1A) SH3BP2 (PH1A) PH Domain Binding selectivity CTH3(PH1A)Akt3(PH1A)PLCd1(PH1A) RalGPS2(PH1A) Hapip1(PH1A) PI(3,4,5)P 3 PI(3,4,)P 2 PI(3,4,5)P 3 PI(4,5)P 2 PI(3,4)P 2 PI(4,5)P 2 PI(3,4,5)P 3 PI(3,4)P 2 Sphingosine-1-phosphate PtdIns(3,4)P 2 PtdIns(3,5)P 2 PtdIns(4,5)P 2 PtdIns(3,4,5)P 3 Phosphatidic acid Phosphatidylserine Blank Lysophosphatidic acid Lysophosphatidylcholine Phosphatidylinositol(PtdIns) PtdIns(3)P PtdIns(4)P PtdIns(5)P Phosphatidylethanolamine Phosphatidylcholine Lipid Binding Categories: PI(3,4,5)P 3 CTH3(PH1A), Myo10(PH1A), ITK(PH1A), H056(PH2A), EtOHD4(PH1A), APS(PH1A), Afap(PH1A), TEC(PH1A) PI(3,4)P 2 and PI(3,4,5)P 3 Gab1(PH1A), Gab2(PH1A), Bam32(PH1A), CTH2(PH1A), IRS-1(PH1A), Osbp13(PH1A), Plek(PH1A), TNFidp(PH1A), Akt2(PH1A), Akt3(PH1A), Akt1(PH1A), LL5(PH1A), Arl61(PH1A), BCRa(PH1a) PI(4,5)P 2 and PI(3,4,5)P 3 PLCd1(PH1A), Spnb2(PH1A), RalGPS2*(PH1A), Centb5(PH1A), Cnk2(PH1A) PI(3,4)P 2 Plek2(PH2A), Hapip1(PH1A) Results of Lipid Binding Assay Of the 127 PH domains tested, 29 show strong specificity in binding to PIP molecules. We placed them in categories based on the phosphoinositides that they bind with the highest affinity. A representative example for each of the PIP-binding classes is shown in the figure panels above. Each of these classes (with the exception of the one shown in the final panel, included specific binding to PI(3,4,5)P 3 Assay for Lipid-Binding Specificity of PH domains The PH domains of many proteins bind to specific phosphoinositides (PIPs). We developed a lipid- blot assay to learn more about the binding specificity of the murine PH domains that we have cloned. In the assay, YFP-tagged PH domain constructs were transfected into 293 cells. Cell lysates were then incubated with nylon membranes bearing a series of specific phosphoinositide spots (below). The YFP-PH domain constructs that had bound to the membrane were then detected using an anti-YFP antibody. We screened 127 PH domains to determine their lipid binding properties. The results of the assays are presented below.