Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation.
Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser144, catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains.
Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California; Department of Orthopedics, The Third Affiliated Hospital Of Southern Medical University, Guangzhou, China.
Canjun
C
Zeng
Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California.
Helen
H
Goodluck
Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California; Department of Medicine, Loma Linda University, Loma Linda, California.
Xuezhong
X
Qin
Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California; Department of Orthopedics, The Third Xiangya Hosptial, Central South University, Changsha, Hunan, China.
Bo
B
Liu
Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California; Department of Medicine, Loma Linda University, Loma Linda, California.
Subburaman
S
Mohan
Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California; Department of Medicine, Loma Linda University, Loma Linda, California; Weirong.xing@va.gov.
Weirong
W
Xing
D000818:Animals; D017089:Ankyrin Repeat; D001862:Bone Resorption / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D009479:Neuropeptides / Q000378:metabolism*; D010010:Osteoclasts / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D053245:RANK Ligand / Q000494:pharmacology; D000067996:RAW 264.7 Cells; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
C115491:Arhgap31 protein, mouse; D020690:GTPase-Activating Proteins; D009479:Neuropeptides; D053245:RANK Ligand; C484602:Rac1 protein, mouse; C505689:Tnfsf11 protein, mouse; C585046:Lrrk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D020830:rac1 GTP-Binding Protein
Lrrk1, bone resorption, osteoclast, RAC1, Cdc42, protein kinase, protein phosphorylation