Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector‐binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans ‐Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild‐type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29‐mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2‐mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.

There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.

ERK5 regulates nonlytic expulsion of live pathogens from phagocytes to limit dissemination of infections.

Focal adhesions are complex multi-molecular structures that link the actin cytoskeleton to the extracellular matrix through integrin adhesion receptors and play a key role in regulation of many cellular functions. LAR (also known as PTPRF) is a receptor protein tyrosine phosphatase that regulates PDGF signalling and localises to focal adhesions. We have observed that loss of LAR phosphatase activity in mouse embryonic fibroblasts results in reduced numbers of focal adhesions and decreased adhesion to fibronectin. To understand how LAR regulates cell adhesion we used phosphoproteomic data, comparing global phosphorylation events in wild-type and LAR phosphatase-deficient cells, to analyse differential kinase activity. Kinase prediction analysis of LAR-regulated phosphosites identified a node of cytoskeleton- and adhesion-related proteins centred on cyclin-dependent kinase-1 (CDK1). We found that loss of LAR activity resulted in reduced activity of CDK1, and that CDK1 activity was required for LAR-mediated focal adhesion complex formation. We also established that LAR regulates CDK1 activity through c-Abl and Akt family proteins. In summary, we have identified a new role for a receptor protein tyrosine phosphatase in regulating CDK1 activity and hence cell adhesion to the extracellular matrix.

Fibroblast Growth Factor (FGF) dependent signalling is frequently activated in cancer by a variety of different mechanisms. However, the downstream signal transduction pathways involved are poorly characterised. Here a quantitative differential phosphoproteomics approach, SILAC, is applied to identify FGF-regulated phosphorylation events in two triple- negative breast tumour cell lines, MFM223 and SUM52, that exhibit amplified expression of FGF receptor 2 (FGFR2) and are dependent on continued FGFR2 signalling for cell viability. Comparative Gene Ontology proteome analysis revealed that SUM52 cells were enriched in proteins associated with cell metabolism and MFM223 cells enriched in proteins associated with cell adhesion and migration. FGFR2 inhibition by SU5402 impacts a significant fraction of the observed phosphoproteome of these cells. This study expands the known landscape of FGF signalling and identifies many new targets for functional investigation. FGF signalling pathways are found to be flexible in architecture as both shared, and divergent, responses to inhibition of FGFR2 kinase activity in the canonical RAF/MAPK/ERK/RSK and PI3K/AKT/PDK/mTOR/S6K pathways are identified. Inhibition of phosphorylation-dependent negative-feedback pathways is observed, defining mechanisms of intrinsic resistance to FGFR2 inhibition. These findings have implications for the therapeutic application of FGFR inhibitors as they identify both common and divergent responses in cells harbouring the same genetic lesion and pathways of drug resistance.