Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities
Authors: James A Mobley
Archer D Smith
Andrew B West
Philip J Webber
Saurabh Sen
Matthew B Renfrow
Journal: Journal of Molecular Biology (2011)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: LRRK2; autophosphorylation; GTPase domain; kinase activity; Parkinson disease
Affiliations: Department of Neurology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Abstract:  The leucine-rich repeat kinase 2 (LRRK2) protein has both guanosine triphosphatase (GTPase) and kinase activities, and mutation in either enzymatic domain can cause late-onset Parkinson disease. Nucleotide binding in the GTPase domain may be required for kinase activity, and residues in the GTPase domain are potential sites for autophosphorylation, suggesting a complex mechanism of intrinsic regulation. To further define the effects of LRRK2 autophosphorylation, we applied a technique optimal for detection of protein phosphorylation, electron transfer dissociation, and identified autophosphorylation events exclusively nearby the nucleotide binding pocket in the GTPase domain. Parkinson-disease-linked mutations alter kinase activity but did not alter autophosphorylation site specificity or sites of phosphorylation in a robust in vitro substrate myelin basic protein. Amino acid substitutions in the GTPase domain have large effects on kinase activity, as insertion of the GTPase-associated R1441C pathogenic mutation together with the G2019S kinase domain mutation resulted in a multiplicative increase (‚à?-fold) in activity. Removal of a conserved autophosphorylation site (T1503) by mutation to an alanine residue resulted in greatly decreased GTP-binding and kinase activities. While autophosphorylation likely serves to potentiate kinase activity, we find that oligomerization and loss of the active dimer species occur in an ATP- and autophosphorylation-independent manner. LRRK2 autophosphorylation sites are overall robustly protected from dephosphorylation in vitro, suggesting tight control over activity in vivo. We developed highly specific antibodies targeting pT1503 but failed to detect endogenous autophosphorylation in protein derived from transgenic mice and cell lines. LRRK2 activity in vivo is unlikely to be constitutive but rather refined to specific responses. 

Retracted: No

PubPeer comments:
 Figure 1 appears to show one band twice (after stretching horizontally), but they seem to be labeled differently (G2019S mutant and WT LRRK2). Could the authors please clarify? ***  , 
 #1 Dear *Actinopolyspora Biskrensis*, A co-author of this study recently , 
 Dear *Actinopolyspora Biskrensis* A co-author recently informed me as to the existence of this post. Thank you for your inquiry and helping to correct the literature. On reviewing the original scans from the silver-stained gels, the  WT-LRRK2 label in part of Figure 1D is incorrect and should be labeled  G2019S-LRRK2 . I paste below the silver stains. Figure 1D came about through addressing reviewer inquiries as to what happens at later time points in the kinase reaction published in Figure 1A, and where the LRRK2 protein might emerge in other regions of the gel than the crop shown in Figure 1A. The figure legend is accurate as is, as well as the corresponding text. As I am the corresponding author, I will contact the journal to see if an erratum is recommended or allowed. Unfortunately, someone has incorrectly entered my email address on this website, and my current valid email address appears locked, I have no idea what an access token is to submit a comment, and I am unable to establish an account that seems to be required by this website to post non-anonymously. Sincerely, Andy West  , 
 #3 Thank you for responding and addressing the concern. As to the email address, I may have been the culprit when I posted the concerns about the papers. I merely used the email address published in the papers, instead of first confirming your current affiliation. You may find other papers by searching by author name: https: pubpeer.com search?q=authors%3A%22Andrew+B.+West%22 , 
 #4 Great!, Thank you for fixing the link and the email issue! I can successfully log in now to the site. I see the issue posted with the duplicated microscopy image in the 2012 J. Neuro paper and we will respond to that very shortly. Sincerely, Andy ,