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First published online 4 December 2002
doi: 10.1242/jcs.00242

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CaM kinase II mediates norepinephrine-induced translocation of cytosolic phospholipase A₂ to the nuclear envelope

Department of Pharmacology and Centers for Connective Tissue Diseases and Vascular Biology, College of Medicine, The University of Tennessee Center for Health Sciences, Memphis, TN 38163, USA

View larger version (37K): [in a new window]   Fig. 1. cPLA2 is localized to the nuclear envelope in sub-confluent and confluent cells in response to norepinephrine (NE) and ionomycin (ION) in VSMCs. Sub-confluent (70% confluent) or confluent (100% confluent) VSMCs from rabbit aorta were stimulated with NE (10 µM), ION (1 µM) or their vehicles (V) for 10 minutes in the presence of 1.8 mM Ca2+. (A) cPLA2 was detected using a monoclonal antibody specific for cPLA2 and a biotinylated second antibody recognized by Texas-Red—streptavidin. Immunofluorescent staining was visualized by confocal microscopy (100x magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope was quantified using the NIH Image 1.62 program as described in Materials and Methods (n=5). *Value significantly different from the corresponding value obtained in the presence of V (P<0.05).

View larger version (15K): [in a new window]   Fig. 2. CaM inhibitors block NE-induced cPLA2 translocation to the nuclear envelope. VSMCs were treated with CaM inhibitors calmidazolium (CLMD, 1 µM), E6-berbamine (E6-B, 10 µM), W-7 (10 µM) or their vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells treated with 10 µM NE or its vehicle (V) and visualized by confocal microscopy (100x magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with V of NE (P<0.05).

View larger version (16K): [in a new window]   Fig. 3. NE-induced translocation of cPLA2 to the nuclear envelope depends on CaMKII. VSMCs were treated with the CaMKII inhibitor KN-93 (10 µM) or its vehicle (VEH), or CaMKII antisense oligonucleotide (5 µM) or its vehicle (lipofectamine; LIPO, 2 mg ml-1). (A) cPLA2 translocation to the nuclear envelope in cells exposed to 10 µM NE or its vehicle (V) was visualized by confocal microscopy (100x magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with V of NE (P<0.05).

View larger version (15K): [in a new window]   Fig. 4. Ionomycin (ION)-induced translocation of cPLA2 to the nuclear envelope depends on CaM and CaMKII. VSMCs were treated with CaM inhibitor W-7 (10 µM) CaMKII inhibitor KN-93 (10 µM) or their vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells exposed to 1 µM ION or its vehicle (V) was visualized by confocal microscopy (100x magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with V of ION (P<0.05).

View larger version (48K): [in a new window]   Fig. 5. NE- and ionomycin (ION)-induced CaMKII activation is attenuated by inhibitors of CaM and CaMKII, and not their structural analogues. VSMCs were treated with NE (10 µM) (A,C), ionomycin (1 µM) (B,D) or vehicle (VEH) in the presence of inhibitors of CaM (10 µM W-7) (A,B), CaMKII (10 µM KN-93) (C,D), their corresponding structural analogues (10 µM W-5, 10 µM KN-92) or their vehicles (V) as described in Materials and Methods. CaMKII activity was determined by immunoblot analysis using a polyclonal antibody against active CaMKII (phospho-CaMKII). The figure shows a representative blot and the densitometric analysis of three experiments performed with each inhibitor and their vehicles on different batches of cells grown in 100 mm tissue culture dishes. The density of CaMKII phosphorylation was quantified using NIH Image 1.62 (n=3). *Value significantly different from the corresponding value obtained with V of NE (A,C) or ION (B,D) (P<0.05).

View larger version (41K): [in a new window]   Fig. 6. Inhibitors of CaM and CaMKII do not change intracellular Ca2+ levels but block NE and ionomycin (ION)-induced cPLA2 phosphorylation. VSMCs were treated with inhibitors of CaM (10 µM W-7) and CaMKII (10 µM KN-93) or their vehicle (VEH) as described in Materials and Methods. (A) Effect of inhibitors of CaM (10 µM W-7) and CaMKII (10 µM KN-93) or vehicle (VEH) on cytosolic Ca2+ ([Ca]i) was measured using fura-2 in the presence of NE (10 µM), ION (1 µM) or vehicle (V). The figure shows a representative of three experiments performed with each agonist and vehicle, and in the presence of different inhibitors on different batches of cells grown on coverslips. (B) Phosphorylation of cPLA2 in response to NE (10 µM), ION (1 µM) or vehicle (V) in the presence (1.8 mM) or absence of extracellular Ca2+. (C,D) Phosphorylation of cPLA2 in response to NE (10 µM) and ION (1 µM) in the presence or absence of inhibitors of CaM (10 µM W-7), CaMKII (10 µM KN-93). (E,F) Phosphorylation of cPLA2 in response to NE (10 µM) and ION (1 µM) in the presence or absence of the corresponding structural analogues of W-7 and KN-93 (10 µM W-5; 10 µM KN-92). Phosphorylation of cPLA2 was determined by incorporation of 32P and detected by autoradiography as described in Materials and Methods. The figure shows a representative autoradiogram and the densitometric analysis from three experiments repeated with each agonist and the vehicle in the presence and absence of each inhibitor performed in different batches of cells grown in 100 mm tissue culture dishes. The density of cPLA2 phosphorylation was quantified using NIH Image 1.62 (n=3). *Value significantly different from the corresponding value obtained in the absence of Ca2+ (B), in the presence of V of NE (C,E) or ION (D,F) (P<0.05).

View larger version (30K): [in a new window]   Fig. 7. NE-induced translocation of cPLA2 to the nuclear envelope depends on phosphorylation. VSMCs were treated with okadaic acid (OA; 10 µM), alkaline phosphatase (AP; 0.5 Unit ml-1; loaded by reversible permeabilization with ß-escin), OA in combination with AP (OA+AP) or vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells treated with 10 µM NE or its vehicle (V) was visualized by confocal microscopy (100x magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from that obtained with V of NE (P<0.05). (C) cPLA2 activity in VSMCs treated with OA, AP, OA+AP or VEH as described in Materials and Methods (n=5). (D) Phosphorylation of cPLA2 as determined by 32P incorporation in cells treated with OA, AP, OA+AP or VEH. A representative of three experiments performed with each agent or its vehicle on different batches of cells grown in 100 mm tissue culture dishes. (E) Density of cPLA2 phosphorylation was quantified using NIH Image 1.62 (densitometric analysis of D).

View larger version (30K): [in a new window]   Fig. 8. NE-induced translocation of cPLA2 to the nuclear envelope depends on its phosphorylation and not its activity. VSMCs were treated with inhibitors of cPLA2 activity arachidonyltrifluoromethyl ketone (AACOCF3; 10 µM) and methyl arachidonyl fluorophosphonate (MAFP; 10 µM) or their vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells treated with 10 µM NE or its vehicle (V) was visualized by confocal microscopy (100x magnification). (B) Density of fluorescence around the nuclear envelope was quantified (n=5). *Value significantly different from that obtained with V of NE (P<0.05). (C) cPLA2 activity in VSMCs treated with AACOCF3, MAFP or VEH in the presence of NE (10 µM) or its vehicle (V) as described in Materials and Methods (n=5). (D) Phosphorylation of cPLA2 as determined by 32P incorporation in VSMCs treated with AACOCF3, MAFP or their vehicle (VEH) and treated with NE (10 µM) or its vehicle (V). The figures shows a representative of three experiments performed with each agent and its vehicle on different batches of cells grown in 100 mm tissue culture dishes. (E) Density of cPLA2 phosphorylation was quantified using NIH Image 1.62 (densitometric analysis of D).

View larger version (36K): [in a new window]   Fig. 9. Phosphorylation-dependent gel shift of cPLA2 (A) and localization of phosphorylated recombinant cPLA2 to the nuclear envelope in cells reversibly permeabilized with ß-escin (B,C). (A) Recombinant cPLA2 unphosphorylated (UP), phosphorylated (P) by purified CaMKII or after dephosphorylation (DP) by alkaline phosphatase (0.5 U ml-1) was separated on 10% SDS-PAGE and analysed for gel shift as described in Materials and Methods. (B) VSMCs were reversibly permeabilized with ß-escin to introduce cPLA2 (0.5 µg) that was unphosphorylated, phosphorylated by CaMKII or dephosphorylated by AP (0.5 Unit ml-1) and detected by immunofluorescence staining. Phosphorylated, but not unphosphorylated or dephosphorylated, cPLA2 translocated to the nuclear envelope (representative of three experiments). In nonpermeabilized cells, unphosphorylated, phosphorylated and dephosphorylated cPLA2 did not translocate to the nuclear envelope. (C) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with unphosphorylated (UP) cPLA2 in permeabilized cells (P<0.05). (D) Unphosphorylated, phosphorylated and dephosphorylated cPLA2 (0.5 µg) conjugated with fluorescence-tagged Alexa 488 was introduced in VSMCs reversibly permeabilized with ß-escin. The figure shows a representative of three experiments. Phosphorylated, but not unphosphorylated or dephosphorylated, cPLA2 accumulated around the nuclear envelope.

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