(A) Kinase inactive LRRK1[D1409A, 20-2015] (200 nM) was incubated ± PKCα (400 nM) in the presence of MgATP. Reactions were terminated after 30 min with SDS-sample buffer and reactions resolved by SDS-polyacrylamide electrophoresis and gel stained by Coomassie blue. (B) The gel bands containing LRRK1 were digested with mixture of trypsin and Lys-C and the resultant peptides analyzed by Electron Activated Dissociation on a ZenoTOF 7600 mass spectrometer without phospho-peptide enrichment. The annotated MS/MS spectra of the three key PKC phosphorylated phosphosites encompassing phosphorylated Thr1061, phosphorylated Ser1064 and doubly phosphorylated Ser1074 and Thr1075 are shown. These sites were only detected in the LRRK1 samples phosphorylated with PKCα. Annotated MS/MS spectra of key PKC regulated phosphosites. MS/MS spectra of pT1061 and pS1064 were identified from HCD fragmentation acquired on Orbitrap Exploris 240 MS platform and the coverage of b and y ion series are denoted by red and blue color text, respectively. MS/MS spectrum of a dual phosphorylated peptide of pS1074 and pT1075 sites were identified from EAD fragmentation acquired on Sciex Zeno-TOF 7600 platform. The EAD fragmentation enables accurate site localization and the coverage for c and z ion series were denoted with blue and red color text. (C) Upper panel displays the location within the COR_B domain arrangement of the Thr1061, Ser1064, Ser1074 and Thr1075 PKCα phosphorylation sites on the domain structure of LRRK1. Lower panels show the amino acid sequences encompassing the PKCα phosphorylation sites and their evolutionary conservation score from 1 (least conserved) to 9 (most conserved) of each residue determined calculated the Consurf server ((https://consurf.tau.ac.il/) [53]. Sequences of LRRK1 homologs were obtained from OrthoDB (https://www.orthodb.org/) [54] and alignment of sequences was performed using the MAFFT server (https://www.ebi.ac.uk/Tools/msa/mafft/) [55]. Subsequently, alignment is inserted into the Consurf server, with the sequence for human LRRK1 indicated as the query sequence (Taxonomy code 9606) and conservation scores are determined. Lower panel displays the conservation score scales utilized by the Cosurf server. Residues lying at the −5, −3 and +1 position of the Thr1075 phosphorylation site that confer optimal specificity for PKC isoform phosphorylation and marked with an asterisks (D) Sequence alignment of LRRK1 and LRRK2 surrounding the LRRK1 PKC phosphorylation sites showing that amino acid sequence across this region is not conserved between LRRK1 and LRRK2.