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-COR_A-COR_B 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 COR_B 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:

1. LRRK1
2. Tubulin
3. Rab7A (Total)

 

Figure 1A Channel 800.tif

Top to bottom:

1. pT202/Y204 ERK1/2
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:

1. Tubulin
2. Rab7A (Total)

 

Figure 1B Channel 800.tif

Top to bottom:

1. pT202/Y204 ERK1/2
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:

1. LRRK2 (Total)
2. Rab10 (Total)
3. 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:

1. LRRK1
2. Tubulin
3. Rab7A (Total)

 

Figure 1D Channel 800.tif

Top to bottom:

1. pT202/Y204 ERK1/2
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:

1. LRRK1
2. Rab7A (Total)

 

Figure 2A GFP IP Kinase Assay Channel 800.tif

pS72 Rab7A

 

Figure 2B

Figure 2B Lysates Channel 700.tif

Top to bottom:

1. LRRK1
2. Tubulin
3. 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:

1. LRRK1
2. 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:

1. LRRK1
2. Tubulin
3. Rab7A (Total)

 

Figure 3B Lysates Channel 800.tif

Top to bottom:

1. pT202/Y204 ERK1/2
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:

1. Tubulin
2. Rab7A (Total)

 

Figure 3C Lysates Channel 700.tif

Top to bottom:

1. LRRK1
2. pT202/Y204 ERK1/2
3. pS72 Rab7A

 

Figure 3D IP Kinase Assay Channel 700.tif

Rab7A (Total)

 

Figure 3D GFP IP Kinase Assay Channel 800.tif

Top to bottom:

1. LRRK1
2. 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:

1. LRRK1 (Total)
2. Rab7A (Total)

 

Figure 4C Channel 800.tif

Top to bottom:

1. pSer-PKC Substrate
2. pS72 Rab7A

 

Figure 4C Merge.tif

Top to bottom:

1. Merge of pSer-PKC substrates in green (channel 800), and total LRRK1 in red (channel 700)
2. 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

1. LRRK1
2. PKCα (Not used in final panel)
3. Rab7A (Total)

 

Figure 5A Channel 800.tif

Top to bottom

1. PKCα
2. 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

1. LRRK1
2. PKCα (Not used in final panel)
3. Rab7A (Total)

 

Figure 5B Channel 800.tif

Top to bottom

1. PKCα
2. 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

1. Top left: LRRK1 (Gel 1)
2. Top right: LRRK1 (Gel 2)
3. Bottom left: Rab7A (Total) (Gel 1)
4. Bottom left: Rab7A (Total) (Gel 2)

 

Figure 7A Channel 800.tif

1. Left: pS72 Rab7A (Gel 1)
2. 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

1. LRRK1
2. 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

1. LRRK1
2. Tubulin (Not used in final panel)
3. Rab7A (Total)

 

Figure 8A Channel 700 GAPDH.tif

GAPDH

 

Figure 8A Channel 800.tif

Top to bottom

1. Na-K ATPase
2. pT202/Y204 ERK1/2
3. 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

1. LRRK1
2. PKCα
3. 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

1. LRRK1
2. PKCα
3. 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 bottom panel.

 

Figure 8C, top panel

All named files below present .lsm files with LRRK1 stained in the green channel (488) and PKCα in the red channel (594)

F8C - DOX - PMA 1.lsm, F8C - DOX - PMA 2.lsm, F8C - DOX - PMA 3.lsm, F8C + DOX - PMA 1.lsm, F8C + DOX - PMA 2.lsm, F8C + DOX - PMA 3.lsm, F8C + DOX - PMA 4.lsm, F8C + DOX - PMA 5.lsm, F8C + DOX - PMA 6.lsm, F8C + DOX + PMA 1.lsm, F8C + DOX + PMA 2.lsm, F8C + DOX + PMA 3.lsm, F8C + DOX + PMA 4.lsm, F8C + DOX + PMA 5.lsm, F8C + DOX + PMA 6.lsm

 

SFigure 6

SFigure 6, left panel

All named files below present .lsm files with LRRK1 stained in the green channel (488) and Na-K ATPase in the red channel (594)

SF6 - DOX - PMA 1.lsm, SF6 - DOX - PMA 2.lsm, SF6 - DOX - PMA 3.lsm, SF6 + DOX - PMA 1.lsm, SF6 + DOX - PMA 2.lsm, SF6 + DOX - PMA 3.lsm, SF6 + DOX - PMA 4.lsm, SF6 + DOX - PMA 5.lsm, SF6 + DOX - PMA 6.lsm, SF6 + DOX + PMA 1.lsm, SF6 + DOX + PMA 2.lsm, SF6 + DOX + PMA 3.lsm, SF6 + DOX + PMA 4.lsm, SF6 + DOX + PMA 5.lsm, SF6 + DOX + PMA 6.lsm

 

SFigure 6, right panel (LRRK1, Na-K ATPase colocalization quantification)

Microscopy images outlined above (and in left panel) were used for quantification in right panel.

 

SFigure 7, top panel

All named files below present .lsm files with LRRK1 stained in the green channel (488) and Na-K ATPase in the red channel (594)

 

SF7 WT + PMA 1.lsm, SF7 WT + PMA 2.lsm, SF7 WT + PMA 3.lsm, SF7 WT + PMA 4.lsm, SF7 WT + PMA 5.lsm, SF7 WT + PMA 6.lsm, SF7 3Ala + PMA 1.lsm, SF7 3Ala + PMA 2.lsm, SF7 3Ala + PMA 3.lsm, SF7 3Ala + PMA 4.lsm, SF7 3Ala + PMA 5.lsm, SF7 3Ala + PMA 6.lsm, SF7 3Glu + PMA 1.lsm, SF7 3Glu + PMA 2.lsm, SF7 3Glu + PMA 3.lsm, SF7 3Glu + PMA 4.lsm, SF7 3Glu + PMA 5.lsm, SF7 3Glu + PMA 6.lsm, SF7 WT - PMA 1.lsm, SF7 WT - PMA 2.lsm, SF7 WT - PMA 3.lsm, SF7 WT - PMA 4.lsm, SF7 WT - PMA 5.lsm, SF7 WT - PMA 6.lsm, SF7 3Ala - PMA 1.lsm, SF7 3Ala - PMA 2.lsm, SF7 3Ala - PMA 3.lsm, SF7 3Ala - PMA 4.lsm, SF7 3Ala - PMA 5.lsm, SF7 3Ala - PMA 6.lsm, SF7 3Glu - PMA 1.lsm, SF7 3Glu - PMA 2.lsm, SF7 3Glu - PMA 3.lsm, SF7 3Glu - PMA 4.lsm, SF7 3Glu - PMA 5.lsm, SF7 3Glu - PMA 6.lsm

 

SFigure 7, bottom panel (LRRK1, Na-K ATPase colocalization quantification)

Microscopy images outlined above (and in top panel) were used for quantification in bottom panel.

 

Instant Blue-stained Gels

Figure 4

Figure 4A and 4C Stained Gels.tif

Instant Blue-stained gels from ³²P-ATP kinase assays performed in Figure 4A and 4B

 

Figure 4B Stained Gel.tif

Scanned gel of PKC isoforms employed in kinase assays for 4A and 4C

 

Figure 5

Figure 5C Stained Gel.tif

Instant Blue-stained gels from ³²P-ATP kinase assays performed in Figure 4A and 4B

 

SFigure 2

SFigure 2 Stained Gel.tif

Instant Blue-stained gels from ³²P-ATP kinase assays performed in SFigure 2

 

Autoradiograph Films

Figure 4:

Figure 4A Autoradiograph.tif

Autoradiograph from ³²P-ATP kinase assay performed in Figure 4A

 

Figure 4C Autoradiograph.tif

Autoradiograph from ³²P-ATP kinase assay performed in Figure 4B

 

Figure 5C Autoradiograph.tif

Autoradiograph from ³²P-ATP kinase assay performed in Figure 5C

 

SFigure 2 Autoradiograph.tif

Autoradiograph from ³²P-ATP kinase assay performed in SFigure 2

 

Graphpad files:

Figure 3A Quantification.pzfx

Graphpad Prism file containing the data shown in Figure 3A.

 

Figure 5A and B Quantification.pzfx

Graphpad Prism file containing the data shown in Figure 5A and B.

 

Figure 7A and B Quantification.pzfx

Graphpad Prism file containing the data shown in Figure 7A and B.

 

Figure 8B Quantification.pzfx

Graphpad Prism file containing the data shown in Figure 8B.

 

SFigure 1 Quantification.pzfx

Graphpad Prism file containing the data shown in SFigure 1.

 

SFigure 2 Quantification.pzfx

Graphpad Prism file containing the data shown in SFigure 2.

 

SFigure 3A and B Quantification.pzfx

Graphpad Prism file containing the data shown in SFigure 3A and B.

 

SFigure 4 Quantification.pzfx

Graphpad Prism file containing the data shown in SFigure 4.

 

SFigure 6 Quantification.pzfx

Graphpad Prism file containing the data shown in SFigure 6.

 

SFigure 7 Quantification.pzfx

Graphpad Prism file containing the data shown in SFigure 7.

 

Excel files:

Figure 3A Quantification.xlsx

Excel file containing the immunoblotting data quantitation (performed using ImageStudio) shown in Figure 3A.

 

Figure 5A and B Quantification.xlsx

Excel file containing the immunoblotting data quantitation (performed using ImageStudio) shown in Figure 5A and B.

 

Figure 7A and B Quantification.xlsx

Excel file containing the immunoblotting data quantitation (performed using ImageStudio) shown in Figure 7A and B.

 

Figure 8B Quantification.xlsx

Excel file containing the colocalization data quantitation (performed using JaCoP plug-in in ImageJ) shown in Figure 8B, left panel.

 

SFigure 1 Quantification.xlsx

Excel file containing the immunoblotting data quantitation (performed using ImageStudio) shown in Figure 1B.

 

SFigure 2 Quantification.xlsx

Excel file containing the scintillation counts (performed using QuantaSmart) shown in SFigure 2.

 

SFigure 3A and B Quantification.xlsx

Excel file containing the immunoblotting data quantitation (performed using ImageStudio) shown in SFigure 3A and B.

 

SFigure 4 Quantification.xlsx

Excel file containing the immunoblotting data quantitation (performed using ImageStudio) shown in SFigure 4.

 

SFigure 6 Quantification.xlsx

Excel file containing the colocalization data quantitation (performed using JaCoP plug-in in ImageJ) shown in SFigure 6, left panel.

 

SFigure 7 Quantification.xlsx

Excel file containing the colocalization data quantitation (performed using JaCoP plug-in in ImageJ) shown in SFigure 7, top panel.