Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Summary

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with ³²P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Abstract

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling^1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease^3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with ³²P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing ³²P-orthophosphate. The ³²P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (³²P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Introduction

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain paralogs which share a similar domain organization. Both proteins encode a GTPase sequence akin to the Ras family of GTPases (Ras of Complex Proteins, or ROC) as well as a C-terminal of ROC domain (COR), effectively classifying both proteins to the ROCO protein family^5,6. N-terminal of the ROC-COR domain tandem, both proteins encode a leucine-rich repeat domain as well as an ankyrin-like domain, while only LRRK2 encodes an extra armadillo domein^6-8. C-terminal of ROC-COR, both proteins share a serine-threonine kinase domain while only LRRK2 encodes a WD40 domain in the C-termin....

Protocol

The present protocol uses radioactive ³²P-labeled orthophosphate to follow cellular phosphorylation of LRRK2. It is important to bear in mind that all operations with radioactive reagents should be performed using appropriate protective measures to minimize exposure of radioactive radiation to the operator and the environment. Compounds containing isotopes that emit ionizing radiation can be harmful to human health and strict licensing and regulations at an institutional and national level control their use. The experiments in this protocol were carried out following training in open source radiation use at Katholieke Universiteit Leuven (KU Leuven) and fol....

Results

In order to compare overall phosphorylation levels of LRRK1 and LRRK2 in cells, 3xflag tagged LRRK1 and LRRK2 were expressed in HEK293T cells¹⁵. Cells were cultured in 6-well plates and labeled with ³²P and analyzed as described above in the protocol text. Figure 1 shows representative results for metabolic labeling of LRRK1 and LRRK2 in HEK293T cells. Radioactive phosphate incorporation is observed for both LRRK1 and LRRK2. Upon quantification of the ³²P levels normalize.......

Discussion

This paper provides a basic protocol for assaying general phosphorylation level of LRRK1 and LRRK2 in cell lines using metabolic labeling with ³²P-orthophosphate. The overall strategy is straightforward. Affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing ³²P-orthophosphate. The ³²P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively lab.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphorus-32 Radionuclide, 1 mCi, buffer disodiumphosphate in 1 ml water	Perkin Elmer	NEX011001MC
Dulbecco’s Modified Eagle Medium (D-MEM) (1X), liquid (high glucose)	Invitrogen	11971-025	This medium contains no phosphates
Anti Flag M2 affiinty gel	Sigma	A2220	For an equivalent product with red colored gel (useful to more easily visualize the beads), use cat. No. F2426.
Extra thick blotting filter	Bio-Rad	1703965
Ponceau S solution	Sigma	P7170