Journal: The Journal of Biological Chemistry

Article Title: Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

doi: 10.1074/jbc.RA118.001779

Figure Lengend Snippet: Rap1b interacts with CAP1. A, two-hybrid isolation of a C-terminal fragment of human CAP1. The activation domain fusion plasmid expressing the C terminus of hCAP1 was cotransformed with empty vector pGBKT7 (Vector) or bait plasmid expressing G12V-Rap1b (G12V) and plated on selection medium lacking Trp, Leu, His, and Ade (QDO; top) versus medium lacking only Trp and Leu (−LT; bottom). B, direct CAP1–Rap1b interaction. Immobilized GST or GST-Rap1b proteins (Coomassie Blue stain shown in lower panel) were incubated with [35S]methionine-labeled in vitro translated CAP1. After extensive washes, samples were resolved by SDS-PAGE. Once dried, radioactivity was visualized by fluorography using a phosphorimaging system (representative image of n = 2). C, in vivo Rap1b and CAP1 interaction. Empty vector (myc-V) or pCMV-myc-CAP1 was cotransfected with HA-Rap1b in HEK293T cells. After 48 h, cell lysates (normalized for equal HA-Rap1b signal) were immunoprecipitated with an anti-myc antibody (9E10), and the presence of Rap1b in the immunocomplex was assessed by Western blotting with an HA-specific antibody. D, in vivo Rap1b and CAP1 interaction. HEK293T cells were transfected with myc-CAP1 plasmid along with GST empty vector or GST-Rap1b mammalian expression plasmids. After 48 h, cell lysates (normalized for equal myc-CAP1 signal) were pulled down with GSH-Sepharose beads. The presence of CAP1 in the complex was assessed by Western blotting with a myc-specific antibody. A representative experiment (n = 3) is shown for C and D. E, colocalization of CAP1 and Rap1b. PCCL3 thyroid follicular cells stably expressing GFP-Rap1b were stained with anti-CAP1 antibodies, and fluorescence microscopy was used to assess intracellular colocalization of CAP1 (red) and Rap1b (green) (representative image; n = 3).

Article Snippet: Anti-CAP1 antibody was from Proteintech.

Techniques: Isolation, Activation Assay, Plasmid Preparation, Expressing, Selection, Staining, Incubation, Labeling, In Vitro, SDS Page, Radioactivity, In Vivo, Immunoprecipitation, Western Blot, Transfection, Stable Transfection, Fluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

doi: 10.1074/jbc.RA118.001779

Figure Lengend Snippet: CAP1 is not a Rap1b effector protein. A, nucleotide-independent Rap1–CAP1 interaction. Lysates expressing HA-Rap1b were loaded in vitro with GDP or GTPγS, and a binding assay was performed upon incubation with myc-CAP1 immobilized on beads. B, Rap1 activation was monitored by GST-RalGDS-RBD pulldown assay. C, HA-CAP1 interacted equally with WT, constitutively active (G12V), and dominant negative (S17N) Rap1 proteins. D, effector domain (ED) mutations in switch I did not affect the interaction between CAP1 and Rap1b. Representative experiments (n = 3) are shown. V, empty vector.

Article Snippet: Anti-CAP1 antibody was from Proteintech.

Techniques: Expressing, In Vitro, Binding Assay, Incubation, Activation Assay, Dominant Negative Mutation, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

doi: 10.1074/jbc.RA118.001779

Figure Lengend Snippet: Rap1 interacts with the C-CAP domain. A, scheme of the CAP1 constructs used in this study. AC, adenylyl cyclase; PR, proline-rich; D, dimerization; FL, full length. B, both GST-N-CAP1 and GST-C-CAP1 failed to interact with Rap1b. The top panel shows purified GST-CAP1 fragments (Coomassie); the bottom panel shows associated Rap1b (HA) after GST-CAP1 pulldown from HA-Rap1b-G12V–expressing cells. C, C-CAP1 dimer formation from X-ray crystal structure (Protein Data Bank code 1K8F). D, C-terminal deletion, myc-C-CAPΔ (aa 319–448), restores binding to immobilized His-Rap1. Representative experiments (n = 3) are shown for B and D.

Article Snippet: Anti-CAP1 antibody was from Proteintech.

Techniques: Construct, Purification, Expressing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

doi: 10.1074/jbc.RA118.001779

Figure Lengend Snippet: Rap1 isoprenylation modulates interaction with CAP1. A, Rap1 lysates from HEK293T cells (i.e. HA-Rap1b-G12V, HA-Rap1b-G12V-GGLL, and HA-Rap1b-G12V-CVLS) were incubated with Ni-NTA-agarose–prebound His-C-CAPΔ for 1 h at 4 °C. After washes, associated proteins were resolved by SDS-PAGE and blotted with anti-HA antibody. B, HA-Rap1b and HA-Ras lysates from HEK293T cells were incubated with Ni-NTA-agarose–prebound His-C-CAP and His-C-CAPΔ. His pulldown was performed as above. Associated proteins were revealed by anti-HA antibody (top panel). His-C-CAP and His-C-CAPΔ total proteins were revealed by anti-His tag antibody (bottom panel). Representative experiments (n = 3) are shown for A and B. C, purified Rap1 was in vitro isoprenylated with purified GGTase I utilizing NBD-FPP (top) as substrate. Total proteins were separated by SDS-PAGE (bottom panel; Coomassie), and fluorescent isoprenylated Rap1 proteins (His-Rap1*) were visualized under UV light (top panel). This experiment was repeated twice with similar results. D, immobilized in vitro isoprenylated (S179D-GG) and control His-Rap1-S179D (S179D) were incubated with normalized myc-C-CAPΔ lysates (Input lanes). The top panel shows the binding results as revealed by anti-myc antibody. The bottom panel shows quantitation of these blots upon densitometric analysis via ImageJ. Curves are expressed as a function of normalized lysate volume. A representative experiment (n = 3) is shown. E, MST analysis of in vitro labeled His-C-CAPΔ-NT647 with purified (Rap1) and in vitro prenylated Rap1 (Rap1-gg) used as titrants. F, MST analysis of labeled Rap1b HVR (OG-HVR) and in vitro prenylated (OG-HVR-gg) peptides and purified His-C-CAPΔ as titrant. G, MST analysis of purified GFP-Rap1b HVR (GFP-C-ter) and control GFP proteins used as probes against purified His-C-CAPΔ as titrant. H, MST analysis of in vitro prenylated (GFP-C-ter-gg) as probe against purified His-C-CAPΔ as titrant. I, MST analysis of the effect of deltarasin titrant on the binding of OG-HVR-GG to C-CAPΔ (conditions equivalent to ∼80% maximum based on F). Error bars for all MST studies represent mean ± S.E. (n = 3), and data analyses were performed using NanoTemper Analysis software.

Article Snippet: Anti-CAP1 antibody was from Proteintech.

Techniques: Incubation, SDS Page, Purification, In Vitro, Control, Binding Assay, Quantitation Assay, Labeling, Software

Journal: The Journal of Biological Chemistry

Article Title: Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

doi: 10.1074/jbc.RA118.001779

Figure Lengend Snippet: Rap1 Ser179 phosphorylation modulates interaction in cells but not in vitro. A, HEK293T cells were co-transfected with GST-Rap1 or GST control and epitope-tagged myc-CAP1 or myc-C-CAPΔ. Upon forskolin stimulation (20 μm, 20min), GST pulldown assays were performed, and associated proteins assessed by Western blots with specific antibodies. B, PKA inhibitor H89 (20 μm) blocked pSer179-GST-Rap1 phosphorylation and stabilized the GST-Rap1–CAP1 interaction. C, The negative effect of forskolin on GST-Rap1–CAP1 interaction can be mimicked by phosphomimetic GST-Rap1-S179D and is lost in phospho-deficient GST-Rap1-S179A. Representative experiments (n = 3) shown in A–C. D, MST analysis of labeled His-C-CAPΔ-NT647, purified Rap1 (Rap-WT and Rap-S179D), and in vitro prenylated (Rap-WT-GG and Rap-S179D-GG) proteins as titrant. Error bars for all MST studies represent mean ± S.E. (n = 3), and data analyses were performed using NanoTemper Analysis software.

Article Snippet: Anti-CAP1 antibody was from Proteintech.

Techniques: Phospho-proteomics, In Vitro, Transfection, Control, Western Blot, Labeling, Purification, Software

Journal: The Journal of Biological Chemistry

Article Title: Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

doi: 10.1074/jbc.RA118.001779

Figure Lengend Snippet: CAP1 depletion alters Rap1 localization. A, CAP1 staining (green channel) in samples of PCCL3 cells transfected with control sh-V (sh-VEC) or sh-CAP1 for 72 h. Transfected cells (red channel) in the field are shown with * in the green channel. B, GFP-Rap1–expressing PCCL3 cells were transfected with sh-CAP1 or sh-V control plasmids. 72 h posttransfection GFP-Rap1 intracellular distribution was assessed by confocal microscopy. Representative fields are shown in A and B. C, quantification of GFP-Rap1 membrane delocalization upon CAP1 depletion. PM/total pixel intensity ratio (green channel) was determined in random red+ cells from experiment shown in B as described under “Experimental procedures.” Data were analyzed, and significance was tested using a two-tailed Student's t test (α level was defined as 0.05). ***, p < 0.0001. All data points are shown. Lines indicate the mean value.

Article Snippet: Anti-CAP1 antibody was from Proteintech.

Techniques: Staining, Transfection, Control, Expressing, Confocal Microscopy, Membrane, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

doi: 10.1074/jbc.RA118.001779

Figure Lengend Snippet: MD simulation and models for lipidated and unmodified peptides and for deltarasin inhibitor. Interactions of RKKSSC-Rap1b-GerGer (A) and -Far (B) prenylated peptides with the N-terminal opening of C-CAP after 5 ns of all-atom dynamics simulation are shown. Both peptides are phosphorylated on Ser179. Although the GG lipid tail is maintained along nearly the entire length of the tunnel, the Far-modified peptide substantially slides out of the tunnel, consistent with weaker binding experimentally (the Far group is initially ∼10 Å shorter than GerGer). Several salt bridges exist between peptide Lys/Arg residues and C-CAP Asp/Glu residues side chains at the N-terminal mouth. In the case of unprocessed peptide and deltarasin inhibitor, initial structures after minimization are shown (C and D). This peptide may be stabilized by the nonpolar side chains in the tunnel (labeled in C), whereas the carbon rings of the inhibitor deltarasin may be stabilized by Phe/Trp and Ile upon initial insertion into N-terminal C-CAP as shown (D). Note that D shows the CAP1 tunnel entrance from the N-terminal side, different from the side-on view of the other panels (A–C).

Article Snippet: Anti-CAP1 antibody was from Proteintech.

Techniques: Modification, Binding Assay, Labeling