A single image through the bottom of the cells is shown. high molecular weight complexes. Separating the Sec14p domain from the spectrin-like repeats eliminates the ability of Kal7 to cause this response. The isolated Sec14p domain binds PIP2(3,5) and PIP3, but does not alter cell morphology. We conclude that the Sec14p and N-terminal spectrin-like domains of Kalirin play critical roles in distinguishing the actions of full-length and -Kalirin. fluorescence assays. For cell based assays, activation of Rac in pEAK Rapid cells transfected with Kalirin plasmids was analyzed using GST-effector protein binding assay kits (Upstate Biosystems, Lake Placid, NY). Lysates prepared in MLB buffer (25 mM HEPES, 150 mM NaCl, 1% Nonidet P40, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, pH 7.5) with 0.3 mg/ml phenylmethyl sulfonyl fluoride (PMSF), 1.0 mM sodium vanadate, and protease inhibitors [19] were incubated with glutathione-agarose beads containing immobilized Pak-CRIB domain (residues 67C150; 10.0 g) and unbound protein was removed with 3 washes of MLB buffer. Extract (50 l) was incubated with 10 mM EDTA and 0.1 mM GTPS (positive control) or 1 mM GDP (negative control) for 20 min at 30C, chilled and brought to 100 mM MgCl2 prior to incubation with GST-effector protein bound to glutathione-agarose beads. Transfected Kalirin proteins and bound Rac (Transduction Laboratories; “type”:”entrez-nucleotide”,”attrs”:”text”:”R56220″,”term_id”:”826326″,”term_text”:”R56220″R56220; 1:1000) were analyzed by Western blot. For fluorescence based GEF assays, the activity of purified Kalirin proteins was assayed by following the release of the methylanthraniloyl analog of GDP (GDP-MANT) from loaded GST-Rac1 with modifications of a previously described assay [20,21]. GST- Rac1 expressed in was purified using glutathione Sepharose [1], dialyzed against 50 mM HEPES, 100 mM NaCl, 1 mM DTT, 1 D-(+)-Xylose mM EDTA, pH 7.6, to remove any bound nucleotide, and then dialyzed against 50 mM HEPES, Rabbit polyclonal to AKT2 100 mM NaCl, 1 mM DTT. His-tagged Kalirin proteins were purified from transfected pEAK-Rapid cells using His-bind resin as D-(+)-Xylose described by the manufacturer (Novagen, Madison, WI), with elution in buffer containing 300 mM imidazole. Before each assay, the dialyzed GST-Rho protein (5C67 M) was loaded with GDP-MANT (50C100 M) in a volume of 80 l. Reactions were supplemented to 10 mM MgCl2 and unbound GDP-MANT was removed using a G50 NICK? column (Pharmacia) equilibrated and eluted in reaction buffer containing 10 mM MgCl2. Fluorescence was measured by excitation at 355 nm and recording emission at 460 nm using a Wallac Victor2 1420 Multilabel 96-well plate reader. Reactions were initiated by adding GTP to 800 M and starting the reaction with 20 l of Kalirin in reaction buffer. Reaction rates were determined by subtraction of the intrinsic rate of loss of fluorescence (reaction lacking enzyme). Subcellular Fractionation and Gel Filtration Parietal cortex from adult rat brain was subjected to subcellular fractionation as described [1,3]; the efficacy of the separation was verified using antisera to PSD95, synaptophysin, calnexin, NMDAR1 and glutamic acid decarboxylase. To obtain cytosol for gel D-(+)-Xylose filtration, adult rat cerebral cortex was homogenized in 320 mM sucrose, 10 mM Tris HCl, 3 mM MgCl2, 1 mM EDTA, pH 7.0 and centrifuged at 435,000 g for 20 min; aliquots (1 mg protein) of the soluble fraction were applied to the gel filtration column. Extracts of transfected pEAK Rapid cells prepared using TMT as described above were also applied to the column. Samples were analyzed on a 1.5 17 cm column of Sephacryl S-400 equilibrated and eluted with 20 mM Hepes, 100 mM NaCl, 0.05% TX-100, pH 7.0. Bovine serum albumin (1 mg) and phenol red (to mark the total volume, Vt) were added to each lysate as internal standards. The column was calibrated using blue dextran (V0), thyroglobulin, catalase, bovine serum albumin, ovalbumin and cytochrome c; the void volume (V0) was 0.32 Vt and BSA eluted at 0.54+0.01 (VBSA/Vt). For unknowns, the elution positions of BSA and D-(+)-Xylose phenol red were determined by monitoring A280 and A560; elution positions are expressed as VX/Vt. Oligomerization of Kalirin GST-Kal7, GST-KalSpec(4C7), GST-KalSpec(4C6) and GST-KalSpec5 bound to glutathione agarose equilibrated with phosphate buffered saline were cleaved with thrombin (1 mg GST-fusion protein; 10 units thrombin); released protein was recovered and pooled with a subsequent wash. After dialysis into 0.1 M NaHCO3/0.5 M NaCl, recombinant protein was linked to 0.5 ml Affi-Gel 15 (BioRad); unreacted sites were blocked by incubation with 0.1 M ethanolamine.