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First published online 15 July 2008
doi: 10.1242/jcs.016089

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Regulation of Kalirin by Cdk5

¹ Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3401, USA
² Endocrine Unit, Massachusetts General Hospital, Boston, MA 02115, USA

View larger version (73K): [in this window] [in a new window]   Fig. 1. Kalirin-7 produces a Cdk5-dependent response in PC12 cells. (A) Domains of Kalirin-7 and its N-terminal -splice variant; S, Sec14P-like; DH, Dbl homology; P, pleckstrin homology; T1590PAK, sequence of putative Cdk5 phosphorylation site. (B) PC12 cells were fixed 24 hours after transfection with vectors encoding GFP or Kalirin-7. Transfected GFP protein was visualized using fluorescence or Myc antibody (green); filamentous actin was visualized using TRITC-phalloidin (red). White arrowheads indicate protrusions, stars indicate nontransfected cells, arrow indicates nucleus. (C) PC12 cells transfected with dual promoter vector encoding GFP (green) and DN Cdk5 (blue); TRITC-phalloidin (red). GFP-expressing cells always expressed DN Cdk5. (D) PC12 cells cotransfected with vectors encoding Kalirin-7 (Myc; blue) and GFP/DN Cdk5 (GFP, green); TRITC-phalloidin (red). White stars indicate nontransfected cells. (E) Using the GFP or Myc signal to identify cell margins, perimeter and cell roundness were evaluated using SimplePCI software: control (GFP), n=18; Kalirin-7 (Kal7), n=15; Kalirin-7 with DN Cdk5, n=24; P values calculated using the Student's t-test (two-tailed). Data are means ± s.e.m.; #, P<0.001. Scale bars: 10 µm (B-D).

View larger version (36K): [in this window] [in a new window]   Fig. 2. Kalirin-Thr1590 is phosphorylated by Cdk5. (A) GST and His-Myc-tagged proteins used in these studies. (B) Kalirin peptides were incubated with 4 ng recombinant Cdk5-p25 complex and 2 µCi [32P]ATP for 30 minutes at 30°C; cpm incorporated into peptide is on the y-axis. Km and Vmax were determined by varying the concentration of peptide from 3.7-100 µM. (C) Recombinant GST-KGEF1-7end and Kalirin-7 are Cdk5-p25 substrates. Recombinant proteins (2 µg) were incubated without or with 4 ng recombinant Cdk5-p25 complex and 2 µCi [32P]ATP for 30 minutes at 30°C. Following fractionation by SDS-PAGE and transfer to a PVDF membrane, samples were visualized by autoradiography (16 hours) or by Coomassie Brilliant Blue staining; the first lane was loaded with molecular size markers. (D) Lysates of pEAK RAPID cells, PC12 cells and mouse striatum (20 µg) were fractionated by SDS-PAGE; endogenous Cdk5, p35, p39 and PP1 were visualized. (E) Top, synthetic Kalirin peptides were incubated with [32P]ATP in the presence of Cdk5-p35 complex immunoprecipitated from pEAK RAPID cells expressing exogenous Cdk5 and p35 using a p35 antibody; 10 µM roscovitine was included in the indicated assays (black bar). Bottom left, indicated doses of roscovitine were incubated with TPAK peptide. Bottom right, TPAK peptide was incubated with [32P]ATP in the presence of Cdk5-p35 or DN Cdk5-p35 complex immunoprecipitated (p35 antibody) from pEAK RAPID cells. Incorporation of 32P into peptide was quantified by Cerenkov counting. (F) pEAK RAPID cells expressing Myc-Kalirin-7 or parent vector (–) and DN Cdk5 were extracted for immunoprecipitation using spectrin-repeat region antibody. Inputs were analyzed using antibody to Myc or Cdk5. Immunoprecipitated Kalirin-7 was visualized using a ThrPro-P antibody. Expression of DN Cdk5 eliminated the band detected by the ThrPro-P antibody.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Thr1590 plays an essential role in the effects of Kalirin-7 on PC12 cell morphology. (A) PC12 cells expressing Kalirin-7 T/A or Kalirin-7 T/D were examined 24 hours post-transfection. Kalirin-7 was visualized using antibody to Myc (green) and filamentous actin was visualized using TRITC-phalloidin (red). Arrowhead indicates protrusion, stars indicate nontransfected cells, arrows indicate nuclei. (B) Cell perimeter and roundness were quantified as described in Fig. 1: GFP, n=27; Kalirin-7 (Kal7), n=24; Kalirin-7-T/A (Kal7 T/A), n=15; Kalirin-7-T/D (Kal7 T/D), n=24. (C) PC12 cells cotransfected with vectors encoding GFP-DN Cdk5 and either Kalirin-7 T/A or Kalirin-7 T/D were fixed after 24 hours. Exogenous Kalirin was visualized with Myc antibody (blue); DN Cdk5 was identified based on coexpression of GFP; filamentous actin was visualized with TRITC-phalloidin (red). Arrowhead, protrusion; white stars, nontransfected cells; arrows, nuclei. (D) Cell perimeter and cell roundness were evaluated as described in Fig. 1: control GFP, n=18; Kalirin-7-T/D, n=15; Kalirin-7-T/D with DN Cdk5, n=18. P values were calculated using Student's t-test (two-tailed) and bars indicate s.e.m.; #P<0.001; *P<0.05. Scale bars, 10 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 4. The GEF activity and spectrin-repeat region of Kalirin-7 are required for protrusion formation. (A) PC12 cells transfected 24 hours earlier with vector encoding inactive Kalirin-7 ND/AA were fixed and Kalirin-7 was visualized using Myc antibody (green). Filamentous actin was visualized using TRITC-phalloidin (red). No morphological differences were detected in cells expressing Kalirin-7 ND/AA. (B) PC12 cells expressing GFP fusion proteins of constitutively active (CA)-Rac or CA-RhoG were fixed after 24 hours. Fusion proteins were visualized based on GFP fluorescence (green); filamentous actin was visualized with TRITC-phalloidin (red); stars, nontransfected cells. (C) PC12 cells expressing KGEF1-7end or the KGEF1-7end T/A and T/D mutants were visualized with Myc antibody (blue), filamentous actin was visualized simultaneously. Scale bars: 10 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 5. A Kalirin antibody specific for Thr1590-P detects Cdk5 phosphorylation of Kalirin-7 and Kalirin-7. (A) A 96-well plate was coated with 50 ng nonphosphorylated peptide () or 50ng QLPK-phospho-T1590PAKLRNNSK (). Bound antibody was visualized using alkaline phosphatase-conjugated antibody to rabbit IgG and p-nitrophenylphosphate. (B) The specificity of the Thr1590-P Ab was tested using purified GST-KGEF1-7end and GST-KGEF1-7endT/A. Aliquots of each fusion protein (2 µg) were analyzed directly (0) or after incubation with recombinant Cdk5-p25 complex (2 ng or 10 ng) and 1 mM ATP. Upper panel: western blot analysis with Thr1590-P antibody. Lower panel: Coomassie Brilliant Blue stained membrane. (C) pEAK RAPID cells transfected with vectors encoding Kalirin-7 or parent vector were treated with or without 10 µM roscovitine (Ros) for 4 hours before extraction into SDS lysis buffer. Duplicate samples were visualized using antibody to Thr1590-P (upper panel) or Myc (lower panel). (D) Quantification of Thr1590-P signal normalized to Myc signal.

View larger version (52K): [in this window] [in a new window]   Fig. 6. Protein phosphatase 1 rapidly dephosphorylates Kalirin phosphorylated on Thr1590. (A) pEAK RAPID cells transiently transfected with vector encoding Kalirin-7 were treated with phosphatase inhibitors as listed on right for 30 minutes before extraction in SDS lysis buffer. Duplicate aliquots of cell extract were visualized with antibody to Thr1590-P (upper panel) or Myc (lower panel). (B) pEAK RAPID cells transiently transfected with parent vector or vectors encoding Kalirin-7, Kalirin-7 or KGEF1 were treated with or without 25 nM calyculin A for 30 minutes before extraction into SDS lysis buffer. Duplicates of cell extracts were visualized with antibody to Thr1590-P (P-T1590 upper panel) or Myc (lower panel). (C) PC12 cells expressing exogenous Myc Kalirin-7 and striatal cultures expressing endogenous Kalirin-7 were exposed to medium containing 10 µM roscovitine for 4 hours or 25nM calyculin A for 30 minutes (CA); control cells (Con) received vehicle.

View larger version (48K): [in this window] [in a new window]   Fig. 7. The GEF activity of Kalirin is increased following calyculin A treatment of cells. Activation of Rac was assessed using GST-Pak-Crib. (A) pEAK RAPID cells transfected with parent vector or vectors encoding KGEF1-7end or KEGF1-7end T/A were untreated (Con) or treated with 10 µM roscovitine (Ros) for 4 hours or 25nM calyculin A (CA) for 30 minutes. Transfected proteins were detected with Myc antibody (upper); phosphorylated protein was detected with Thr1590-P Ab (second row), and total Rac (third row) was visualized with Rac antibody. GTP-bound Rac was isolated using GST-Pak-Crib resin and quantified by western blot analysis with Rac antibody (bottom). (B,C) pEAK RAPID cells transfected with vectors encoding Kalirin-7 (B) or Kalirin-7 (C) were analyzed as described in A. Control cells were transfected with parent vector. Pooled cell extract incubated with GDP or GTPS, negative and positive controls. (D) Data from three independent assays. Activated Rac in control cells expressing Kalirin-7 (upper) or Kalirin-7 (lower) set to 100%; activated Rac in roscovitine or calyculin-A-treated cells expressed as a percentage of relevant control; P values were calculated using Student's t-test; *P<0.05.

View larger version (26K): [in this window] [in a new window]   Fig. 8. GEF activity of Kalirin is increased by mutation of Thr1590 to Asp. GEF activity was assessed in vitro using GDP-Mant-loaded Rac1 (2 µM). (A) Change in fluorescence over time. Buffer or the proteins indicated (final concentration 20 µM) were added at t=3 minutes. (B) Data from the first 20 minutes converted to a semi-logarithmic plot enabled initial rate calculation. (C) Data from three assays for GEF activities of GST-KGEF1-K7end, KGEF1-K7end T/A and KGEF1-K7end T/D were averaged. Significant differences are indicated; *P<0.05.

View larger version (32K): [in this window] [in a new window]   Fig. 9. Kalirin-7 T/D shows increased solubility compared with Kalirin-7. (A) Cortex from adult rat brain was separated into cytosol, organelle and TX-100-insoluble fractions; 20 µg protein from each fraction was subjected to western blot analysis using antibody specific for Kalirin-7. (B) PC12 cells expressing wild-type Kalirin-7, Kalirin-7 T/A and Kalirin-7 T/D were separated into soluble and insoluble fractions; 10% of the soluble fraction (Sol) and 1% of the resuspended pellet fraction (Pel) was subjected to western blot analysis for Myc. Bar graph shows quantification of data from two independent experiments; error bars indicate range.

View larger version (83K): [in this window] [in a new window]   Fig. 10. Thr1590 of Kalirin-7 plays an essential role in determining the shape of dendritic spines. Cortical neurons from P1 rats were nucleofected on day in vitro (DIV) 1 with vector encoding GFP alone (A) or cotransfected with vectors encoding GFP and Kalirin-7 (B), Kalirin-7 T/A (C) or Kalirin-7 T/D (D). Fixed neurons were immunostained for GFP (left) or Myc (middle) (DIV16). Merged images are shown in panels on the right. Scale bars, 10 µm.

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