Curr Biol. 2008 Feb 12;18(3):168-76. doi: 10.1016/j.cub.2007.12.058.

RACK1 inhibits TRPM6 activity via phosphorylation of the fused alpha-kinase domain.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Cao, G., Thebault, S., van der Wijst, J., van der Kemp, A., Lasonder, E., Bindels, R. J., Hoenderop, J. G.,
["Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands."]
BACKGROUND: The maintenance of the body's Mg(2+) balance is of great importance because of its involvement in numerous enzymatic systems and its intervention in neuromuscular excitability, protein synthesis, and nucleic acid stability. Recently, the transient receptor potential melastatin 6 (TRPM6) was identified as the gatekeeper of active Mg(2+) transport and therefore plays a crucial role in the regulation of Mg(2+) homeostasis. Remarkably, TRPM6 combines a Mg(2+) channel with an alpha-kinase domain whose function remains elusive. RESULTS: Here, we identify the receptor for activated C-kinase 1 (RACK1) as the first regulatory protein of TRPM6 that associates with the alpha-kinase domain. RACK1 and TRPM6 are both present in renal Mg(2+)-transporting distal convoluted tubules. We demonstrate that RACK1 inhibits channel activity in an alpha-kinase activity-dependent manner, whereas small interference (si) RNA-mediated knockdown of RACK1 increases the current. Moreover, threonine(1851) in the alpha-kinase domain was identified as an autophosphorylation site of which the phosphorylation state is essential for the inhibitory effect of RACK1. Importantly, threonine(1851) was crucial for the Mg(2+) sensitivity of TRPM6 autophosphorylation and channel activity. TRPM6 channel activity was less sensitive to Mg(2+) when RACK1 was knocked down by siRNA. Finally, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA prohibited the inhibitory effect of RACK1 on TRPM6 channel activity. CONCLUSIONS: We propose a unique mode of TRPM6 regulation in which the Mg(2+) influx is controlled by RACK1 through its interaction with the alpha-kinase and the phosphorylation state of the threonine(1851) residue.
PMID: 18258429External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Yeast two-hybrid Human 1075-2022 Mouse Kidney cDNA library 18258429
TRPM7 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Inference Prediction 18258429
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vitro validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Yeast two-hybrid Human 1075-2022 Yeast Mouse Full-length Yeast 18258429
TRPM7 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Fusion protein-pull down assay Mouse ơ-kinase domain E. coli Mouse Full-length In vitro translation 18258429
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with endogenous proteins Assay with overexpressed proteins Reference
Cell or tissue Cell or tissue TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Species Region
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Co-immunoprecipitation HEK293 Human Full-length Not specified Not specified 18258429
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Co-immunofluorescence staining Mouse kidney section 18258429
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Fusion protein-pull down assay Mouse 1857-1885 Mouse Not determined 18258429
TRPM7 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Fusion protein-pull down assay Mouse ơ-kinase domain Mouse Not determined 18258429
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Functional consequence Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
TRP channel Interactor Method Post-translational modification Subcellular trafficking Activity Reference
TRPM6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Patch clamp Inhibition 18258429
TRPM7 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 RacK1 Patch clamp Inhibition 18258429
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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