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Science 314 (5796): 122-125

Copyright © 2006 by the American Association for the Advancement of Science

Action of TFII-I Outside the Nucleus as an Inhibitor of Agonist-Induced Calcium Entry

Gabriela Caraveo1, Damian B. van Rossum2, Randen L. Patterson2*, Solomon H. Snyder2,3,4, and Stephen Desiderio1{dagger}

1 Department of Molecular Biology and Genetics, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
2 Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
3 Pharmacology and Molecular Science, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
4 Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.


Figure 1 Fig. 1.. Specific binding of PLC-{gamma} to a TFII-I tyrosine phosphopeptide. (A) Sequences of peptide 73, corresponding to a region spanning the phosphorylation site at Y462, and control peptide 74. (B) Peptide-bound proteins from a Ramos B cell lysate were fractionated by SDS–polyacrylamide gel electrophoresis and detected by silver staining. (C) Large-scale purification yielded a protein of similar size to that in (B) from beads carrying peptide 73, as detected by Coomasie Blue stain. (D) Specific binding of PLC-{gamma}2 to peptide 73 was confirmed by immunoblotting; WCL, whole cell lysate. (E) FLAG-tagged wild-type (wt) and mutant PLC-{gamma}1 proteins were expressed in PC12 cells, and lysates were incubated with peptide beads. Proteins bound to peptide 73 or 74 were detected by immunoblotting. Un, untransfected. (F) PLC-{gamma}2 was immunoprecipitated (Ip) from the Daudi B cell line before and after B cell receptor cross-linking; precipitates were immunoblotted for TFII-I. Minutes of stimulation are indicated at top; 0* indicates F(ab')2 added after lysis. (G) PLC-{gamma}2 was immunoprecipitated (Ippt) from the Daudi or from the K562 hematopoietic cell line, and precipitates were assayed by immunoblotting for TFII-I. IgG, immunoglobulin G. [View Larger Version of this Image (41K GIF file)]
 

Figure 2 Fig. 2.. Suppression of ACE by wild-type or cytosolic TFII-I. (A) PC12 cells were transfected with pMACS Kk. II and siRNA directed against rat TFII-I (Sp) or control (C) siRNA as indicated. Lysates of H2Kk-expressing cells were assayed for TFII-I or tubulin by immunoblotting. (B) After cotransfection with yellow fluorescent protein (YFP) and Sp or C siRNA, PC12 and HEK cells were stimulated with UTP or bradykinin, and [Ca2+]i was measured in YFP-positive cells. Sp siRNA in HEK cells was directed against human TFII-I. Extracellular calcium was present at times indicated (Ca++). (C) PC12 cells were transfected with pMACS Kk. II and Sp siRNA, C siRNA, pCIS2, human TFII-I, or human TFII-I{Delta}NLS, as indicated. H2Kk-expressing cells were assayed for TFII-I or tubulin by immunoblotting. (D) Representative calcium traces of bradykinin-stimulated PC12 cells cotransfected with YFP, C siRNA, and pCIS2; YFP, Sp siRNA, and pCIS2; or YFP, Sp siRNA, and human TFII-I. (E) TFII-I and TFII-I{Delta}NLS were detected in transfected cells by immunofluorescence (rhodamine) and confocal microscopy; nuclei were stained with 4',6'-diamidino-2-phenylindole. (F) Representative calcium traces of bradykinin-stimulated PC12 cells transfected as in (D) or with YFP, Sp siRNA, and human TFII-I{Delta}NLS. [View Larger Version of this Image (38K GIF file)]
 

Figure 3 Fig. 3.. A split PH-like domain of TFII-I interacts with PLC-{gamma}. (A) Sequence of the split PH-like domain identified in figs. S5A and S6. N-terminal (PHNlike) portion, red; C-terminal (PHClike) portion, blue. Bold letters, HLH-like repeat sequence. Arrowhead, tyrosine phosphorylation site. (B) GST fusions to the PH-like domain or the PHClike portion, and GST alone, were tested for binding to membrane-immobilized phospholipids identified at right and in fig. S3. Bound protein was detected by immunoblotting for GST. [View Larger Version of this Image (33K GIF file)]
 

Figure 4 Fig. 4.. Failure of TFII-I({Delta}PHNlike,YY/FF) to suppress ACE. (A) PC12 cells were transfected with a plasmid encoding myc-tagged human (h) TFII-I or myc-tagged TFII-I({Delta}PHNlike,YY/FF) or with pCIS2 and assayed by immunoblotting for Myc. (B) Representative calcium traces of bradykinin-stimulated PC12 cells cotransfected with YFP, control (C) siRNA, and pCIS2; YFP, specific (Sp) siRNA, and pCIS2; YFP, Sp siRNA, and human TFII-I; or YFP, Sp siRNA, and human TFII-I({Delta}PHNlike,YY/FF). (C) Calcium entry, defined as the integral of [Ca++]i over the time interval of 200 to 400 s, was normalized to control. Mean and SEM are plotted for 350 cells from each transfected cell population in (B). Difference between wild type and TFII-I({Delta}PHNlike,YY/FF) estimated as pairwise comparison by using a two-tailed t test; one way analysis of variance (ANOVA) yielded similar results. [View Larger Version of this Image (27K GIF file)]
 

Figure 5 Fig. 5.. TRPC3 accumulation after depletion of TFII-I. (A) PC12 cells were transfected with pMACS Kk. II and specific (Sp) siRNA, control (C) siRNA, pCIS2, and human TFII-I as indicated. H2Kk-expressing cells were isolated and subjected to cell surface biotinylation. Biotinylated protein was immunoprecipitated with streptavidin agarose. Precipitated protein (streptavidin IP) or whole cell lysates (WCL) were assayed by immunoblotting for TRPC3 or the cytosolic control protein heme oxygenase 2 (HO2). (B) Entry of extracellular calcium was measured without agonist treatment in PC12 cells transfected as in Fig. 2C. Extracellular calcium was present for the period indicated by the bar. (C) PC12 cells expressing FLAG-tagged PLC-{gamma}1 under doxycycline control (lanes 2 to 5 and 7 to 10) were transfected with myc-tagged TFII-I, TFII-I{Delta}NLS, or pCIS2 as indicated. Untransfected PC12 cells (Lanes 1 and 6) were examined in parallel. Transfected cells, cultured in the presence (lanes 2 and 7) or absence (lanes 3 to 5 and 8 to 10) of doxycycline, were biotinylated at the cell surface as in (A). Precipitated protein (lanes 1 to 5) or whole cell lysates (lanes 6 to 10) were assayed by immunoblotting for TRPC3 and cytosolic (HO2) or surface (CD71) control proteins. Transfected PLC-{gamma}1 and TFII-I were detected with antibodies to FLAG and myc epitopes, respectively. The histogram indicates the relative ratio of TRPC3 to CD71 in streptavidin precipitates, normalized to the ratio obtained for untransfected PC12 cells. [View Larger Version of this Image (37K GIF file)]
 


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