Leucine-Rich Repeat Kinase 1 Signaling Targets Proteins Critical for Endosome/Lysosome Sorting and Trafficking in...

Global knockout (KO) of the Lrrk1 gene in mice causes severe osteopetrosis because of the failure of osteoclasts to resorb bone. The molecular mechanism of LRRK1 regulation of osteoclast function is not fully understood. Here, we performed a 2D DIGE phosphor-proteomics analysis to identify potential LRRK1 targets in osteoclasts. Splenocytes from Lrrk1 KO and wild-type (WT) mice were differentiated into osteoclasts for protein extraction. Lysates from Lrrk1 KO and WT cells were labeled with Cy3- and Cy5-dye, respectively. Labeled proteins were mixed and analyzed on the same 2D SDS PAGE for protein profiling. The same amounts of cellular protein were also labeled with Cy3-dye and ran on a 2D SDS PAGE. The gels were then stained using Pro-Q^® Diamond Phosphoprotein Gel Stain for phosphoprotein profiling. Differentially phosphorylated protein spots between the two types of cells were collected, digested with trypsin, and identified by mass spectrometry. Seventeen phosphoproteins were identified, six of which are known to be involved in endosome/lysosome sorting, vacuolar protection, and trafficking. While five of these proteins (SNX2, VPS35, VTA1, CFL1, and CTSA) were significantly hypophosphorylated, SNX3 was hyperphosphorylated in LRRK1-deficient osteoclasts. The downregulation of VSP35 and CFL1 phosphorylation in LRRK1-deficient cells was validated by Phos-tag SDS PAGE analysis. Our results indicate that LRRK1 signaling regulates osteoclast function via modulating VPS35 and CFL1 phosphorylation critical for endosome/lysosome trafficking and dynamic cytoskeleton arrangement in osteoclasts.