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Regulatory pathways acting on inositol trisphosphate receptor localization and function

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  1. Regulatory pathways acting oninositol trisphosphate receptor localization and function Jan B. Parys K.U.Leuven Univ. AlbertaEdmonton20-22 July 2004

  2. Intracellular Ca2+ homeostasis Tetrameric structure Genes: 3 Regulation by Ca2+ (Clapham, 1995)

  3. Monomeric type 1 IP3R (IP3R1) N IP3 REGULATORY SITES Ca2+-binding sitesPKA/PKG phosphoryl.ATP binding Caspase-3 cleavage C

  4. Monomeric type 1 IP3R (IP3R1) N IP3 REGULATORY SITES Ca2+-binding sitesPKA/PKG phosphoryl.ATP binding Caspase-3 cleavage C

  5. Monomeric type 1 IP3R (IP3R1) N IP3 REGULATORY SITES Ca2+-binding sitesPKA/PKG phosphoryl.ATP binding Caspase-3 cleavage C


  6. Monomeric type 1 IP3R (IP3R1) N IP3 REGULATORY SITES Ca2+-binding sitesPKA/PKG phosphoryl.ATP binding Caspase-3 cleavage C

  7. CaBP CaM CARP IRBIT CASK and/orsyndecan-2 RACK1 Gβ Cyt c AKAP9 4.1N PP1 PKA PP1 Spectrin-actincytoskeleton HAP1A/Htt ANKYRIN B CHROMOGRANIN A & B SIG-1R Monomeric type 1 IP3R (IP3R1) mGluR 1a, 5 Shank IP3R RyRCa2+ channels HOMER N TRP IP3 C

  8. Regulation of the IP3R by calmodulin • Dynamic regulation of the IP3R byprotein kinase C • Activation of an IP3-independentpathway by IP3R1 cleavage throughcaspase-3

  9. Regulation of the IP3R by calmodulin

  10. Effects of CaM on IP3-induced Ca2+ release: A7r5 (Missiaen et al., 1999)

  11. Effects of CaM on IP3-induced Ca2+ release: 200 μM Ca2++10 μM CaM A7r5 (Missiaen et al., 1999) Cerebellum (Michikawa et al., 1999)

  12. Effects of CaM on IP3-induced Ca2+ release: 200 μM Ca2++10 μM CaM - CaM + CaM A7r5 (Missiaen et al., 1999) Cerebellum (Michikawa et al., 1999) 16HBE14o- (Missiaen et al., 2000)

  13. Effects of CaM on IP3-induced Ca2+ release: Effects of CaM on IP3 binding: 200 μM Ca2++10 μM CaM - CaM + CaM A7r5 (Missiaen et al., 1999) Cerebellum (Michikawa et al., 1999) 16HBE14o- (Missiaen et al., 2000) Sf9 (Cardy & Taylor, 1998)

  14. Effects of CaM on IP3-induced Ca2+ release: Effects of CaM on IP3 binding: 200 μM Ca2++10 μM CaM - CaM + CaM A7r5 (Missiaen et al., 1999) Cerebellum (Michikawa et al., 1999) 16HBE14o- (Missiaen et al., 2000) Lbs-1 Lbs-2 Lbs-3 CaM (μM) Sf9 (Cardy & Taylor, 1998) Lbs-domains (Vanlingen et al., 2000)

  15. Effects of CaM on IP3-induced Ca2+ release: Ca2+ dependent Effects of CaM on IP3 binding: Ca2+ independent 200 μM Ca2++10 μM CaM - CaM + CaM A7r5 (Missiaen et al., 1999) Cerebellum (Michikawa et al., 1999) 16HBE14o- (Missiaen et al., 2000) Lbs-1 Lbs-2 Lbs-3 CaM (μM) Sf9 (Cardy & Taylor, 1998) Lbs-domains (Vanlingen et al., 2000)

  16. CaM Monomeric type 1 IP3R (IP3R1) Low-affinity Ca2+-independent Responsible for effects on IP3 binding High-affinity Ca2+-dependent Mutations ineffective Not in IP3R3 N IP3 C Low-affinity Ca2+-dependent Not in neuronal IP3R1

  17. 1 581 Lbs-1 226 581 Lbs-1 1-225 Cyt1 Cyt2 Detailed analysis of the N-terminal CaM-binding site +HIS +HIS +GST 1 159 154 309 1-5-10 1-5-10 53% IQ 1-5-8-14 76% IQ 70% IQ A C D B E F 1.0 2+ 200 µM free Ca 0.8 • Band-shift experiments on non-denaturing gels • Interaction with dansyl-CaM 1 mM EGTA 0.6 0.4 0.2 0.0 -0.2 A B C D E F

  18. 1 581 Lbs-1 226 581 Lbs-1 1-225 Cyt1 Cyt2 Detailed analysis of the N-terminal CaM-binding site +HIS +HIS +GST 1 159 154 309 1-5-10 1-5-10 53% IQ 1-5-8-14 76% IQ 70% IQ A C D B E F 1.0 200 µM Ca2+1mM EGTA Kd 0.1 μM Kd 1 μM 0.8 0.6 0.4 Intensity loss (1-B/Bo) 0.2 0.0 (Sienaert et al., 2002) -0.2 A B C D E F

  19. Both the B and the E sites are necessary for CaM binding 1 159 A C D E B F control ∆ B ∆ E [3H]IP3 binding CaM - + - + - +

  20. CaM N-CaM C-CaM [3H]IP3 binding 45Ca2+ flux 80 100 60 80 60 40 40 20 2+ Ca 20 45 0 0 control control controle N-CaM C-CaM N+C-CaM CaM Lbs-1 Lbs-1 N CaM Lbs-1 C Ca Lbs-1 N+C CaM Lbs-1 CaM Both the N- and C-terminal parts of CaM are needed

  21. Inhibitory Activatory ??? CaM-like Ca2+-binding proteins (Haeseleer et al., 2002)

  22. CaM CaM 1 159 C A D E B F Binding of CaBP1 to the same site? GST 1-604 GST 1-225 GST 226-604 GST CaBP1

  23. Binding of CaBP1 to the same sitebut only to the domain B GST 1-604 GST 1-225 GST 226-604 GST CaBP1 CaM CaM 1 159 C A D E B F CaBP1

  24. CaBP binding is also Ca2+ independent Ratio of CaBP: peptide B CaBP CaBP 1/1 1/2 1/3 1/4 1/5 1/6 1/8 1/10 Ca2+ EGTA 1.0 Ca2+ EGTA 0.5 Band intensity 0.0 (Nadif Kasri et al.,2004) 0 2 4 6 8 10 Peptide B: CaBP

  25. 600 CaM 400 Ca2+i (nM) 200 CaM1234 0 0 250 500 750 1000 IICR is inhibited in vivo by CaM and CaM1234 … 0.5 µM 1 µM 100 µM ATP Control Time (s)

  26. 1000 800 600 Ca2+i (nM) sCaBP lCaBP 400 200 0 0 200 400 600 800 1000 Time (s) … and by both short and long CaBP1 100µM 1µM 0.5µM ATP control (Nadif Kasri et al., 2004)

  27. 1000 800 600 Ca2+i (nM) sCaBP lCaBP 400 CaBP134 200 0 0 200 400 600 800 1000 Time (s) … and by both short and long CaBP1and also by the Ca2+-insensitive CaBP1134 100µM 1µM 0.5µM ATP control (Nadif Kasri et al., 2004)

  28. Suramin interacts with the CaM-binding sites In the presence of B In the presence of E CaM Suramin 0 100 µM 0 100 µM aa 226-581 aa 1-581 +suramin (Nadif Kasri et al., in press)

  29. Calmodulin is not the Ca2+ sensor of the IP3R L15 fibroblasts (Nadif Kasri et al., in press)

  30. - Ca2+ + Ca2+ Change in way that CaM, CaBP, … interacts Model: IP3R structure is dependent on Ca2+ (Hamada et al., 2003)

  31. Dynamics concerning the intracellular localization of the IP3R • Dynamic regulation of the IP3R byprotein kinase C • Activation of an IP3-independentpathway by IP3R1 cleavage throughcaspase-3

  32. Localization of IP3R1 and IP3R3in A7r5 smooth-muscle cells IP3R1 IP3R3 (Vermassen et al., 2003)

  33. AVP > 1h PLC activationIP3-ester ThapsigarginCPA [Ca2+]cyt Redistribution of IP3R1 after prolonged stimulation Resting cells + AVP (Vermassen et al., 2003)

  34. ER-targetedEYFP PDI SERCA Control AVP Structure of the endoplasmic reticulum

  35. Factors participating in IP3R redistribution PKC activator OAG induction Staurosporine inhibitionPKC inhibitors Bisindolylmaleimide inhibition Gö-6876 inhibition Drugs acting on Nocodazole inhibitionmicrotubuli Taxol inhibition Action on vesicle Brefeldin A inductiontransport Cooling to 15 ºC inhibition

  36. TG  Ca2+ OAG  PKC Do other IP3R isoformsalso redistribute in a similar way? IP3R3 in16HBE14o-cells Control Agonist (ATP)

  37. IP3R redistribution is dependent on the cell status Agonist( ) IP3Rredistribution

  38. PKC-mediated phosphorylation of IP3R1 [32P]ATP Anti-Ser-P

  39. [32P]ATP Anti-IP3R PKC-mediated phosphorylation of IP3R1and IP3R3 [32P]ATP Anti-Ser-P

  40. PKA-mediated phosphorylation of IP3R1stimulates PKC-mediated phosphorylation Anti-Ser-P

  41. Ca2+ inhibits PKC-mediated phosphorylation of IP3R1

  42. Ca2+ and calmodulin differentiallyregulate PKC-mediated phosphorylation ofIP3R1 and IP3R3

  43. 0.2 µg/ml αCT 0.4 µg/ml αCT Control 273 kDa 225 kDa 210 kDa 130 kDa Blot strippedand reprobed 40 kDa Anti-(1829-1848) Ab Determination of the PKC phosphorylationsites on IP3R1 and IP3R3 Purified IP3R1 273 kDa 225 kDa 210 kDa 130 kDa 40 kDa Phosphorylatedfragments

  44. CaM Low-affinity Ca2+-independent Responsible for effects on IP3 binding High-affinity Ca2+-dependent Mutations ineffective Not in IP3R3 PKA/PKG phosphoryl. Low-affinity Ca2+-dependent Not in neuronal IP3R1 Monomeric type 1 IP3R (IP3R1) N IP3 C

  45. Potential physiological role ofPKC-mediated phosphorylation of IP3R1

  46. ACTIVATION INHIBITION PKC Ca2+ Ca2+ CaM PP - function ? - interaction ? Potential physiological role ofPKC-mediated phosphorylation of IP3R1 CaMorPreincubation

  47. Dynamics concerning the intracellular localization of the IP3R • Dynamic regulation of the IP3R byprotein kinase C • Activation of an IP3-independentpathway by IP3R1 cleavage throughcaspase-3

  48. ACT. IP3R 95K During apoptosis, IP3R1 is cleavedto a 95 kDa C-terminal fragment For expression in IP3R ko cells: IN.

  49. N C N C (Δ1-1891)IP3R1 expression does not reduce the amount of releasable Ca2+ from the ER N C in the presence of Ca2+ex

  50. Caspase-3 mediated cleavage of IP3R1leads to an increase in ... Caspase-3 activity Apoptosis