IGF Receptors

(B) Extracts of HEK293 cells transfected for 48 h with [wt]LRRK2 were operate on a 6% polyacrylamide gel, immunoblotted with the antibody against LRRK2-MID (lower panel) and reprobed with an antibody against the HA tag (upper panel)

(B) Extracts of HEK293 cells transfected for 48 h with [wt]LRRK2 were operate on a 6% polyacrylamide gel, immunoblotted with the antibody against LRRK2-MID (lower panel) and reprobed with an antibody against the HA tag (upper panel). encodes a large multi-domain (see Fig. 1A) ROCO protein [14] characterized by the presence of a Ras-of-Complex (Roc) domain name with high similarity to small GTPases and a C-terminal-of-Roc (COR) domain name involved in dimerization [15,16]. Additionally, LRRK2 has a second catalytic domain name, a serine/threonine kinase with sequence homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) [17]. These key signal transduction molecules are involved in Epithalon a vast number of cellular functions such as proliferation, differentiation and survival and have been shown to Goat monoclonal antibody to Goat antiMouse IgG HRP. play a crucial role in regulating neural functions [18,19]. Open in a separate window Fig. 1 Expression and distribution of LRRK2. (A) A LRRK2-specific rabbit polyclonal antibody was raised against the region 800C1000 amino acids (MID) between the ANK and LRR domains of human LRRK2. Location of the mutations used in the study is usually shown in red. R1441C in the COR domain name and G2019S in the kinase domain name are PD-associated mutations, whereas K1906N is usually a synthetic mutant with an impaired kinase activity. (B) Extracts of HEK293 cells transfected for 48 h with [wt]LRRK2 were run on a 6% polyacrylamide gel, immunoblotted with the antibody against LRRK2-MID (lower panel) and reprobed with an antibody against the HA tag (upper panel). LRRK2 protein is usually detected at approximately 280 kDa by both HA-specific and LRRK2-specific antibodies. The LRRK2 antibody detected also endogenous LRRK2 in extracts from mock-transfected cells. (For interpretation of the recommendations to colour in this physique legend, the reader is referred to the web version of this article.) More than 50 LRRK2 variants have been identified in PD patients. To date R1441C/G, Y1699C, G2019S and I2020T have been proven to be pathogenic. Mutation G2019S has been regarded as the most common cause of dominant inherited as well as sporadic PD [20] and has been shown to significantly increase both auto- and MBP-phosphorylation compared to [wt]LRRK2 [21]. This effect together with the kinase-dependent toxicity of some of the mutants [22,23] suggests a mechanism of toxic gain-of-function, probably related to deregulation of LRRK2 kinase activity. Increasing evidence indicates that this major pathology associated to mutations in and particularly to G2019S [13] is usually SNCA aggregation, pointing towards a causative link between mutations and SNCA pathology. However, it is unclear if Epithalon LRRK2 directly binds to and phosphorylates SNCA [21,24]. MAPKKKs are enzymes often acting as initiators of signaling cascades that ultimately lead to transcriptional regulation [25]. Thus, we hypothesized a potential regulation of through a MAPK signaling cascade initiated by LRRK2. We systematically analyzed the activation state of the three MAPK modules (extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), p38MAPK) after LRRK2 over-expression in HEK293 cells. Our results indicate that LRRK2 is usually a functional serine/threonine kinase that selectively activates the ERK pathway, but not the p38MAPK and JNK pathways, in a kinase-dependent manner. Interestingly, LRRK2 mutations R1441C and G2019S caused a significant delay of ERK activation. Activation of the ERK pathway by LRRK2 stimulated endogenous transcription, resulting in increased expression. Our results indicate a Epithalon functional link between the two autosomal-dominant PD genes and through the activation of a specific kinase cascade that might be relevant in the development of new therapies for the treatment of both familial and sporadic PD. 2. Materials and methods 2.1. Vectors and constructs The 7.58 kb LRRK2 gene was amplified with a high fidelity PCR System (Roche) by using four complementary fragments from human brain (BD Biosciences Clontech) and cerebellum cDNA libraries (Dr. K. Kaupmann, Novartis). Full-length cDNA was generated by subcloning the fragments in the mammalian expression vector pCI (Promega) using the three unique Epithalon internal restriction sites AfeI, NdeI and ClaI. The C-terminal HA tag, as well as the missense mutations R1441C, K1906N and G2019S were inserted using the QuikChange? site-directed mutagenesis kit (Stratagene) using wild-type plasmid pCI LRRK2 as template for the PCR reactions. The entire CMV promoter and Epithalon coding sequences were verified by automated sequencing. 2.2. Cell culture and inhibitors To generate LRRK2-inducible cells, 293-FT inducible Flp-In cells (Invitrogen) were used. Integration was achieved by transfection with WT, G2019S or R1441C LRRK2 in combination with the fip recombinase expressed from the pOG44 plasmid. 293-FT cells were selected based on Zeocin (100 g/ml)/hygromycin (200.