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. 2010 Oct 7;5(10):e13191.
doi: 10.1371/journal.pone.0013191.

Signal transduction protein array analysis links LRRK2 to Ste20 kinases and PKC zeta that modulate neuronal plasticity

Susanne Zach  1 , Sandra Felk, Frank Gillardon
Affiliations

Signal transduction protein array analysis links LRRK2 to Ste20 kinases and PKC zeta that modulate neuronal plasticity

Susanne Zach et al. PLoS One. .

Abstract

Background: Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease, however, the underlying pathogenic mechanisms are poorly understood. Several in vitro studies have shown that the most frequent mutation, LRRK2(G2019S), increases kinase activity and impairs neuronal survival. LRRK2 has been linked to the mitogen-activated protein kinase kinase kinase family and the receptor-interacting protein kinases based on sequence similarity within the kinase domain and in vitro substrate phosphorylation.

Methodology/principal findings: We used an unbiased proteomic approach to identify the kinase signaling pathways wherein LRRK2 may be active. By incubation of protein microarrays containing 260 signal transduction proteins we detected four arrayed Ste20 serine/threonine kinase family members (TAOK3, STK3, STK24, STK25) as novel LRRK2 substrates and LRRK2 interacting proteins, respectively. Moreover, we found that protein kinase C (PKC) zeta binds and phosphorylates LRRK2 both in vitro and in vivo.

Conclusions/significance: Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations.

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Conflict of interest statement

Competing Interests: The authors are employees of Boehringer Ingelheim Pharma GmbH & Co KG. Funding does not alter adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Recombinant LRRK2 binds and phosphorylates arrayed serine/threonine kinase 25.
(A) Magnified sections of Panorama Human Protein Function Arrays incubated with GST-tagged LRRK2(G2019S) (amino acids 970-2527) and [γ-33P]ATP (left panel) or buffer and [γ-33P]ATP (right panel). The phosphoscreen image shows an increase in radioactive signal intensity on the LRRK2-treated protein arrays in the quadruplicate spots of serine/threonine kinase 25. (B) Arrays incubated with GST-tagged LRRK2(G2019S) and cold ATP (left panel) or recombinant GST and cold ATP (right panel). GST-tagged LRRK2 bound to spotted serine/threonine kinase 25 is detected by an Alexa 488-conjugated anti-GST antibody. The GST-treated control array is negative. Orange fluorescence of Cy3-labeled duplicate protein spots is visible in the upper left corner which serve as markers for grid alignment during image analysis.
Figure 2
Figure 2. LRRK2 phosphorylates receptor interacting kinase 2 on protein microarrays.
Magnified sections of Panorama Human Protein Function Arrays incubated with LRRK2(G2019S) and [γ-33P]ATP (left panel) or buffer and [γ-33P]ATP (right panel). The quadruplicate spots of receptor interacting kinase 2 exhibit an increased radioactive signal when incubated with recombinant LRRK2(G2019S).
Figure 3
Figure 3. LRRK2 binds to spotted protein kinase C zeta and becomes phosphorylated.
Panorama Human Protein Function Arrays incubated with (A) GST-tagged LRRK2(G2019S) and [γ-33P]ATP (left panel) or buffer and [γ-33P]ATP (right panel), (B) GST-tagged LRRK2(G2019S) and cold ATP or recombinant GST and cold ATP, (C) kinase-inactive GST-tagged LRRK2(D1994A) and [γ-33P]ATP or buffer and [γ-33P]ATP. (A and C) An increase in radioactive signal intensity is visibly on the LRRK2-treated protein arrays in the quadruplicate spots of protein kinase C zeta. (B) GST-tagged LRRK2 bound to spotted protein kinase C zeta is visualized by an Alexa 488-conjugated anti-GST antibody. Control arrays show background signal only. Orange fluorescence of Cy3-labeled duplicate protein spots is visible in the upper left corner which serve as markers for grid alignment during image analysis.
Figure 4
Figure 4. Recombinant protein kinase C zeta phosphorylates a LRRK2 fragment in solution.
(Upper panel) Autoradiogram demonstrating phosphorylation of GST-tagged LRRK2(G2019S) (amino acids 970-2527) (lane 1), GST-tagged wildtype LRRK2 (lane 2) and kinase-inactive GST-tagged LRRK2(D1994A) (lane 3) by recombinant protein kinase C zeta in vitro. Background autophosphorylation of the three kinases in the absence of protein kinase C zeta is shown in lanes 5–7. GST-tagged moesin is not a substrate for protein kinase C zeta excluding GST phosphorylation by protein kinase C zeta (lane 4). (Lower panel) Coomassie Blue protein staining of the gel shows similar amounts of recombinant LRRK2.
Figure 5
Figure 5. Protein kinase C zeta phosphorylates full-length LRRK2 in vitro.
(Upper panel) Autoradiogram showing phosphorylation of full-length, wildtype LRRK2 (lane 2, 4) and full-length, kinase-dead LRRK2(K1906M) (lane 1, 3) by recombinant protein kinase C zeta (lane 1, 2) in vitro. (Lower panel) Coomassie Blue protein staining shows similar amounts of recombinant LRRK2. Two LRRK2 fragments are visible migrating below 250 kDa that are also phosphorylated by protein kinase C zeta.
Figure 6
Figure 6. LRRK2 co-immunoprecipitates with protein kinase C zeta from mouse brain.
Homogenates of C57BL/6 mouse brains were incubated with an anti-protein kinase C zeta antibody (lane 2) or pre-immune serum as control (lane 1). Immuncomplexes were precipitated using protein G-sepharose beads and subjected to gel electrophoresis and immunoblotting using either an anti-LRRK2 antibody (upper panel) or an anti-protein kinase C zeta antibody (lower panel). Representative results are shown for experiments that were repeated two times.
Figure 7
Figure 7. LRRK2 phosphorylates MARKK in vitro.
(Left panel) Autoradiogram showing phosphorylation of recombinant, kinase-inactive MARKK(K57A) by GST-tagged LRRK2(G2019S) (amino acids 970-2527) (lane 2). Kinase-inactive GST-tagged LRRK2(D1994A) was used as negative control (lane 1). Endogenous tubulin-beta that is co-purified with LRRK2 becomes phosphorylated as well. (Right panel) Coomassie Blue protein staining shows similar amounts of recombinant proteins.
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