PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the CORB GTPase domain

Abstract Leucine-rich-repeat-kinase 1 (LRRK1) and its homologue LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause Parkinson’s disease. Previous work suggested that LRRK1 but not LRRK2, is activated via a Protein Kinase C (PKC)-dependent mechanism. Here we demonstrate that phosphorylation and activation of LRRK1 in HEK293 cells is blocked by PKC inhibitors including LXS-196 (Darovasertib), a compound that has entered clinical trials. We show multiple PKC isoforms phosphorylate and activate recombinant LRRK1 in a manner reversed by phosphatase treatment. PKCα phosphorylates LRRK1 at a cluster of conserved residues (Ser1064, Ser1074 and Thr1075) located within a region of the CORB domain that lies at the equivalent region of the LRRK2 DK helix that is reported to stabilize the kinase domain αC-helix in the active conformation. Thr1075 lies within an optimal PKC site phosphorylation motif and its mutation to Ala, blocked PKC-mediated activation of LRRK1. A triple Glu mutation ofSer1064/Ser1074/Thr1075 to mimic phosphorylation, enhanced LRRK1 kinase activity ~3-fold. From analysis of available structures, we postulate that phosphorylation of Ser1064, Ser1074 and Thr1075 could activate LRRK1 by promoting interaction of these residues with the aC-helix on the kinase domain. This study provides fundamental insights into the mechanism controlling LRRK1 activity. LI-COR (.tif files) Figure 1, Figure 1A Figure 1A Channel 700.tif Top to bottom: LRRK1 Tubulin Rab7A (Total) Figure 1A Channel 800.tif Top to bottom: pT202/Y204 ERK1/2 pS72 Rab7A Figure 1A Channel 800 Total ERK1_2.tif ERK1/2 (Total) Figure 1A Channel 800 pSer PKC substrate.tif pSer-PKC Substrate Figure 1B, top panel Figure 1B Channel 700 LRRK1.tif LRRK1 Figure 1B Channel 700 PKC Alpha.tif PKCα Figure 1B Channel 700.tif Top to bottom: Tubulin Rab7A (Total) Figure 1B Channel 800.tif Top to bottom: pT202/Y204 ERK1/2 pS72 Rab7A Figure 1B Channel 800 pSer PKC substrate.tif pSer-PKC Substrate Figure 1C Figure 1C Channel 700 Total Rab7A.tif Rab7A (Total) Figure 1C Channel 700.tif Top to bottom: LRRK2 (Total) Rab10 (Total) Tubulin Figure 1C Channel 800 LRRK1.tif LRRK1 Figure 1C Channel 800 pERK1_2.tif pT202/Y204 ERK1/2 Figure 1C Channel 800 pRab7A.tif pS72 Rab7A Figure 1C Channel 800 High Exposure pRab10.tif pT73 Rab10 Figure 1D Figure 1D Channel 700.tif Top to bottom: LRRK1 Tubulin Rab7A (Total) Figure 1D Channel 800.tif Top to bottom: pT202/Y204 ERK1/2 pS72 Rab7A Figure 1D Channel 800 pSer PKC substrate.tif pSer-PKC Substrate Figure 2, Figure 2A Figure 2A GFP IP Kinase Assay Channel 700.tif Top to bottom: LRRK1 Rab7A (Total) Figure 2A GFP IP Kinase Assay Channel 800.tif pS72 Rab7A Figure 2B Figure 2B Lysates Channel 700.tif Top to bottom: LRRK1 Tubulin Rab7A (Total) Figure 2B Lysates Channel 800.tif pS72 Rab7A Figure 3, Figure 3A, top panel Figure 3A GFP IP Channel 800 PKC Substrate.tif pSer-PKC Substrate Figure 3A GFP IP Kinase Assay Channel 700.tif Top to bottom: LRRK1 Rab7A (Total) Figure 3A GFP IP Kinase Assay Channel 800.tif pS72 Rab7A Figure 3A, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 3A were used for quantification in bottom panel. Figure 3B Figure 3B Lysates Channel 700.tif Top to bottom: LRRK1 Tubulin Rab7A (Total) Figure 3B Lysates Channel 800.tif Top to bottom: pT202/Y204 ERK1/2 pS72 Rab7A Figure 3B Lysates Channel 800 pSer PKC substrate.tif pSer-PKC Substrate Figure 3B Lysates Channel 800 pPKD1.tif pS910 PKD1 Figure 3B Lysates Channel 800 Total PKD1.tif PKD1 (Total) Figure 3C Figure 3C Lysates Channel 700.tif Top to bottom: Tubulin Rab7A (Total) Figure 3C Lysates Channel 700.tif Top to bottom: LRRK1 pT202/Y204 ERK1/2 pS72 Rab7A Figure 3D IP Kinase Assay Channel 700.tif Rab7A (Total) Figure 3D GFP IP Kinase Assay Channel 800.tif Top to bottom: LRRK1 pS72 Rab7A Figure 4, Figure 4A Figure 4A Channel 700.tif Total LRRK1 Figure 4A Channel 800.tif pSer-PKC Substrate Figure 4A Merge.tif Merge of pSer-PKC substrates in green (channel 800), and total LRRK1 in red (channel 700) Figure 4C Figure 4C Channel 700.tif Top to bottom: LRRK1 (Total) Rab7A (Total) Figure 4C Channel 800.tif Top to bottom: pSer-PKC Substrate pS72 Rab7A Figure 4C Merge.tif Top to bottom: Merge of pSer-PKC substrates in green (channel 800), and total LRRK1 in red (channel 700) Merge of pS72 Rab7A substrates in green (channel 800), and total Rab7A in red (channel 700) Figure 5, Figure 5A, top panel Figure 5A Channel 700.tif Top to bottom LRRK1 PKCα (Not used in final panel) Rab7A (Total) Figure 5A Channel 800.tif Top to bottom PKCα pS72 Rab7A Figure 5A, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 5A were used for quantification in bottom panel. Figure 5B, top panel Figure 5B Channel 700.tif Top to bottom LRRK1 PKCα (Not used in final panel) Rab7A (Total) Figure 5B Channel 800.tif Top to bottom PKCα pS72 Rab7A Figure 5B, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 5B were used for quantification in bottom panel. Figure 5C Figure 5C GFP IP Channel 700.tif LRRK1 (Total) Figure 5C GFP IP Channel 800.tif pSer-PKC substrates Figure 5C GFP IP Channel Merge.tif Merge of pSer-PKC substrates in green (channel 800), and total LRRK1 in red (channel 700) Figure 6, Figure 6A Figure 6A Channel 700.tif Scanned gel of kinase assay employing LRRK1 (upper band) and PKCα (lower band) Figure 7, Figure 7A Figure 7A Channel 700.tif Top left: LRRK1 (Gel 1) Top right: LRRK1 (Gel 2) Bottom left: Rab7A (Total) (Gel 1) Bottom left: Rab7A (Total) (Gel 2) Figure 7A Channel 800.tif Left: pS72 Rab7A (Gel 1) Right: pS72 Rab7A (Gel 2) Figure 7A, bottom Panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 7A were used for quantification in bottom panel. Figure 7B Figure 7B Channel 700.tif Top to bottom LRRK1 Rab7A (Total) Figure 7B Channel 800.tif pS72 Rab7A Figure 7A, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of Figure 7A were used for quantification in bottom panel. Figure 8, Figure 8A Figure 8A Channel 700.tif Top to bottom LRRK1 Tubulin (Not used in final panel) Rab7A (Total) Figure 8A Channel 700 GAPDH.tif GAPDH Figure 8A Channel 800.tif Top to bottom Na-K ATPase pT202/Y204 ERK1/2 pS72 Rab7A Figure 8A Channel 800 PKC Alpha.tif PKCα Figure 9 Figure 9.tif Annotated AlphaFold model of LRRK1 SFigure 3, SFigure 3A, top panel SFigure 3A Channel 700 LRRK1.tif LRRK1 SFigure 3A Channel 700 Total Rab7A.tif Rab7A (Total) SFigure 3A Channel 800 PKC Alpha.tif PKCα SFigure 3A Channel 800 pRab7A.tif pS72 Rab7A SFigure 3A, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of SFigure 13A were used for quantification in bottom panel. SFigure 3B, top panel SFigure 3B Channel 700.tif Top to bottom LRRK1 PKCα Rab7A (Total) SFigure 3B Channel 800.tif pS72 Rab7A SFigure 3B, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of SFigure 1B were used for quantification in bottom panel. SFigure 4 SFigure 4 Channel 700.tif Top to bottom LRRK1 PKCα Rab7A (Total) SFigure 4 Channel 800.tif pS72 Rab7A SFigure 4, bottom panel (pRab7A quantification) pS72 Rab7A and blots outlined in top panel of SFigure 2 were used for quantification in bottom panel. Microscopy Images (.lsm files) Figure 8 Figure 8B, top panel All named files below present .lsm files with LRRK1 stained in the green channel (488) and PKCα in the red channel (594) F8B - DOX - PMA 1.lsm, F8B - DOX - PMA 2.lsm, F8B + DOX - PMA 1.lsm, F8B + DOX - PMA 2.lsm, F8B + DOX - PMA 3.lsm, F8B + DOX - PMA 4.lsm, F8B + DOX - PMA 5.lsm, F8B + DOX - PMA 6.lsm, F8B + DOX + PMA 1.lsm, F8B + DOX + PMA 2.lsm, F8B + DOX + PMA 3.lsm, F8B + DOX + PMA 4.lsm, F8B + DOX + PMA 5.lsm, F8B + DOX + PMA 6.lsm Figure 8B, bottom panel (LRRK1, PKCα colocalization quantification) Microscopy images outlined above (and in top panel) were used for quantification in bot

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