TY - JOUR

T1 - Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

AU - Massip, L

AU - Garand, C

AU - Labbé, A

AU - Perreault, E

AU - Turaga, R V N

AU - Bohr, V A

AU - Lebel, M

PY - 2010/3/11

Y1 - 2010/3/11

N2 - Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.

AB - Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.

KW - Blotting, Western

KW - Cell Line, Tumor

KW - Cells, Cultured

KW - DNA Damage

KW - Enzyme Activation

KW - Exodeoxyribonucleases

KW - Fibroblasts

KW - GTP-Binding Proteins

KW - Humans

KW - Neoplasm Proteins

KW - Phosphoproteins

KW - Phosphorylation

KW - Protein Binding

KW - Protein Kinase C

KW - Protein Kinase C-delta

KW - RNA Interference

KW - RecQ Helicases

KW - Receptors, Cell Surface

KW - p38 Mitogen-Activated Protein Kinases

U2 - 10.1038/onc.2009.443

DO - 10.1038/onc.2009.443

M3 - Journal article

C2 - 19966859

VL - 29

SP - 1486

EP - 1497

JO - Oncogene

JF - Oncogene

SN - 0950-9232

IS - 10

ER -