Regulation of the subcellular localization and phosphorylation of the

1. Regulation of the subcellular localization and phosphorylation of the inositol 1,4,5-trisphosphate receptor 18/06/2004

2. DAG α ß  IP3 Ca2+ signals Ca2+ ADP ATP PMCA Ca2+ General introduction RTK PIP2 PIP2 Na+/Ca2+ exchanger GPCR PLC ß PLC α α ß Gq  IP3R Ca2+ ER Ca2+ influx channel SERCA

3. [Ca2+]i time Ca2+ signals → regulation in time and space: “spatio-temporal character” • elementary signals: • blips • puffs • global signals: • oscillations • waves → cell-type dependent and the underlying mechanisms are still unknown PART ONE:Subcellular localization of IP3Rs PART TWO: Differential regulation of IP3R isoforms

4. PART ONE: The regulation of the subcellular localization of IP3Rs in A7r5 smooth muscle cells

5. Overview part one The regulation of the subcellular localization of IP3Rs in A7r5 smooth muscle cells. • Introduction • Subcellular localization of IP3R1 and IP3R3 in A7r5 • Localization of IP3Rs in other cell types • Interaction of IP3Rs with cytoskeletal proteins and protein kinases

6. Ligand binding regulatory channel 1. Introduction • IP3R = intracellular Ca2+-release channel located predominantly • on ER membranes • 3 known isoforms: IP3R1, IP3R2 and IP3R3

7. Distribution of IP3Rs dependent on cell type • Different isoforms can have different localizations in the cell • One isoform can have more than one localization in the cell • Short-term agonist stimulation can lead to clustering of IP3Rs → role of long-term stimulation

8. 3 µM AVP 5 h 2. Subcellular localization of IP3R1 and IP3R3 in A7r5 IP3R1 IP3R3 Control

9. 80 cytoplasmic 70 60 50 Cells (%) 40 30 perinuclear 20 10 0 0 1 2 3 4 5 Time (h) Redistribution occurs mostly during the second hour after addition of AVP and the process is reversible Time course of IP3R1 redistribution

10. 100 90 80 70 Relative fluorescence (%) 60 50 40 30 20 80 10 70 0 60 0 1 2 3 4 5 50 Time (h) Time (h) Cells (%) 40 30 20 10 0 Redistribution of IP3R1 = downregulation? Long term stimulation → downregulation of IP3Rs cytoplasmic perinuclear 0 1 2 3 4 5 IP3R1 Kinetics of both processes are completely different.

11. 80 70 60 50 40 30 20 10 0 Downregulation  redistribution IP3R1 Downregulation → ubiquitin-proteasome pathway Cells with perinuclear IP3R1 (%) control 3 µM AVP 20 µM MG-132 + 3 µM AVP

12. Imipramine PLC ß CPA OAG Gö-6976 + AVP Staurosporine + AVP Imipramine thapsigargin Bisindolylmaleimide + AVP Thapsigargin CPA Staurosporine Bisindolylmaleimide Gö-6976 OAG PKC plays an important role in the redistribution of IP3R1 Factors involved in the redistribution of IP3R1 Vasopressin (AVP) DAG PIP2 V1a α PKC α α ß ß Gq   IP3 IP3R Ca2+ ER AVP Control SERCA

13. PDI SERCA ER structure seems to be unaffected, but proteins involved in Ca2+ signaling redistribute after prolonged stimulation. Localization of ER proteins ER-targeted EYFP Control AVP

14. control AVP OAG microtubuli actin Mechanism of redistribution of IP3R1 a) Cytoskeleton

15. An intact microtubular network is necessary for redistribution of IP3R1

16. Control AVP 14°C AVP 37°C Brefeldin A Redistribution could occur through vesicle trafficking b) Vesicle trafficking % cells with perinuclear IP3R1

17. HeLa 16HBE14o- SH-SY5Y IP3R1 IP3R3 IP3R3 IP3R1 untreated untreated untreated Carbachol (10 µM) Histamine (1 µM) ATP (50 µM) TG (1 µM) TG (1 µM) TG (1 µM) Bar = 10 µM OAG (50 µM) 3. Localization of IP3Rs in other cell types

18. Vimentin Tubulin Zyxin Actin Talin Vinculin Proteins expressed in A7r5: 4. Interaction of IP3Rs with cytoskeletal proteins and protein kinases in A7r5 Restriction of IP3Rs in a specific part of the cell could be due to interactions with cytoskeletal proteins e.g. focal adhesion proteins.

19. IP3R3 interacts with talin and actin, while IP3R1 only interacts with talin. Immunoprecipitation of IP3R1 and IP3R3 with cytoskeletal proteins

20. IP3R3 interacts with the regulatory subunit of PKA. Immunoprecipitation of IP3R1 and IP3R3 with protein kinases Heavy chain IP3R3 antibody Negative control beads Pre-immune serum IP IP3R3 IP IP3R1 Lysate PKA PKC Even after stimulation of cells with OAG during different times, no co-immunoprecipitation of IP3R1 or IP3R3 with PKC was found.

21. SUMMARY PART ONE 1. IP3R1 redistribution in A7r5 cells • IP3R1 and IP3R3 have a different subcellular localization in A7r5 • IP3R1 and SERCA redistribute after prolonged stimulation with AVP • Redistribution  downregulation: 2 independent processes • Activation of PKC is sufficient to induce redistribution • ER structure undergoes no major morphological changes • An intact microtubular network is necessary for redistribution • Process likely occurs through vesicular trafficking

22. 2. Interaction of IP3Rs with cytoskeletal proteins and protein kinases • IP3R3 interacts with actin and talin, while IP3R1 only interacts • with talin • IP3R3 interacts with the regulatory subunit of PKA

23. PART TWO: The regulation of the phosphorylation of IP3R1 and IP3R3 by PKC

24. Overview part two The regulation of the phosphorylation of IP3R1 and IP3R3 by PKC. • Introduction • In vitro phosphorylation of IP3R1 and IP3R3 by PKC • Determination of the phosphorylation site(s) on IP3R1 and IP3R3 • Influence of PKA phosphorylation on PKC phosphorylation • Differential regulation of the PKC phosphorylation by Ca2+ and CaM • Potential role in the negative slope of the bell-shaped effect of Ca2+

25. IP3 IP3R1, IP3R2, IP3R3 IP3R1, IP3R2 CaM Stimulation of IICR ≤ 300 nM ≥ 300 nM [Ca2+]i 1.Introduction Regulation of IP3R activity Ligand binding domain Regulatory domain Channel domain * Ca2+ * CaM → Nael

26. 1.Introduction 1.Introduction Regulation of IP3R activity Regulation of IP3R activity Ligand binding domain Ligand binding domain Regulatory domain Regulatory domain Channel domain Channel domain * Ca2+ * CaM → Nael IP3 IP3 * ATP … * contain several consensus sequences for different kinases.

27. IP3R1: - PKA - PKG - PKC - CaMKII - Fyn and Lyn - Rho kinase - cdc2/cyclin B - ERK IP3R2: - PKA - Rho kinase IP3R3: - PKA - Rho kinase stimulates IICR ? stimulates IICR? ? stimulates IICR stimulates IICR IP3 binding ↑ ? ? no effect on IICR? ? no effect on IICR?

28. 2. In vitro phosphorylation of IP3R1 and IP3R3 by PKC Catalytic subunit of PKC (, , ) IP3Rs purified from Sf9 microsomes on sucrose density gradient and/or CaM-sepharose column → highly purified fraction. IP3R1 32P phospho (Ser) PKC Ab

29. Autoradiography Rbt475 IP3R3 is also phosphorylated, but to a lesser degree

30. 3. Determination of the phosphorylation site(s) on IP3R1 and IP3R3 →controlled proteolysis with -chymotrypsin after in vitro phosph. IP3R1 Yoshikawa et. al., J. Biol. Chem., 1999, 274, 316-327 5 fragments

31. 0.2 µg/ml CT 0.4 µg/ml CT Control 273 kDa 225 kDa 210 kDa 130 kDa 40 kDa Phosphorylated fragments

32. 3. Determination of the phosphorylation site(s) on IP3R1 and IP3R3 →controlled proteolysis with -chymotrypsin after in vitro phosph. IP3R1 Yoshikawa et. al., J. Biol. Chem., 1999, 274, 316-327 Anti-(1829-1848) 5 fragments

33. 0.2 µg/ml CT 0.4 µg/ml CT Control 273 kDa 273 kDa 225 kDa 210 kDa 225 kDa 210 kDa 130 kDa 130 kDa Blot stripped and reprobed 40 kDa 40 kDa Phosphorylated fragments anti-(1829-1848) Ab

34. IVa 40k

35. S1852? IP3R1 S1832? IP3R3 CaCaM mass spectrometry: IP3R1 and IP3R3 performed by Prof. E. Waelkens (Biochemie) • Phosphorylated receptor → SDS-PAGE • In gel digestion • Extraction of proteolytic peptides • Concentration of phosphopeptides • Mass spectrometry

36. Prior phosphorylation with PKA enhances PKC phosphorylation Pathways activating PKA and PKC may have synergistic effects on the potentiation of Ca2+ release by IP3R1. 4. Influence of PKA phosphorylation on PKC phosphorylation IP3R1

37. + Ca2+ (50 µM) - Ca2+ IP3R1 IP3R3 IC50 ≤ 2 µM Ca2+ inhibits PKC phosphorylation 5. Differential regulation of the PKC phosphorylation by Ca2+ and CaM Ca2+

38. CaM CaM1234 CaM1234 CaM inhibits the phosphorylation of IP3R1 CaM

39. CaM Ca2+-dependent kinases and phosphatases may play an important role in the regulation of the IP3R. 6. Potential role in the negative slope of the bell-shaped effect of Ca2+ Stimulation of IICR ≤ 300 nM ≥ 300 nM [Ca2+]i

40. SUMMARY PART TWO • Both IP3R1 and IP3R3 are in vitro phosphorylated by PKC, although • to a different extent. • IP3R1 contains 1 PKC phosphorylation site, probably S1852. • IP3R3 also contains 1 PKC phosphorylation site, probably S1832. • Prior phosphorylation of IP3R1 with PKA enhances PKC-mediated • phosphorylation → pathways involving PKA and PKC may have a • synergistic effect. • Ca2+ and CaM inhibit PKC phosphorylation of IP3R1 • → could play a role in the negative feedback of • Ca2+ and CaM on IP3R function. • IP3R3 phosphorylation is not affected by Ca2+ and CaM.