Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation

Abstract

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.

LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L‐plastin

“…The enriched phospho-peptides were then dried, resuspended in 0.1% formic acid, and directly subjected to LS/MS at the University of California, Riverside, MS core facility. An aliquot of total cellular lysate (30 μg) was separated by 10% NuPAGE for Western blot analysis as described previously (11) .…”

“…Our structure-function studies revealed that ANK, ROC, and the kinase domains of Lrrk1 were essential for the Lrrk1 mediated bone resorption function in OCs (11) . We have also demonstrated that Tyr-527 phosphorylation of c-Src was altered in Lrrk1 deficient OCs (9) .…”

“…An aliquot of total cellular lysate (30 μg) was separated by 10% NuPAGE for Western blot analysis as described previously. 11…”

“…We have also demonstrated that Tyr-527 phosphorylation of c-Src was altered in Lrrk1 deficient OCs (9) . Phosphorylation and activation of Rac1/Cdc42 were also reduced in Lrrk1 KO cells (11) . Because mice with Lrrk1 disruption exhibit a more severe osteopetrosis phenotype than c-Src KO mice and Rac1/Rac2 double KO mice, it can be predicted that Lrrk1 signaling may target other signaling molecules besides the c-Src/Rac1/Cdc42 small GTPase signaling pathways via post-translational modification (12) .…”

“…9 Phosphorylation and activation of Rac1/Cdc42 were also reduced in Lrrk1 KO cells. 11 Because mice with Lrrk1 disruption exhibit a more severe osteopetrosis phenotype than c-Src KO mice and Rac1/Rac2 double KO mice, it can be predicted that Lrrk1 signaling may target other signaling molecules besides the c-Src/Rac1/Cdc42 small GTPase signaling pathways via posttranslational modification. 12 To understand how Lrrk1 regulates formation of F-actin rings and podosomes in OCs, we have examined proteins that are differentially phosphorylated in the wild type (WT) and Lrrk1 deficient OCs by LC/MS.…”