Disease-associated Kinase Research Articles

Impact of protein and small molecule interactions on kinase conformations.

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

Impact of protein and small molecule interactions on kinase conformations

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

Opposing actions of JIP4 and RILPL1 provide antagonistic motor force to dynamically regulate membrane reformation during lysosomal tubulation/sorting driven by LRRK2.

Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. We previously uncovered a process we term LYTL (LYsosomal Tubulation/sorting driven by Leucine-Rich Repeat Kinase 2 [LRRK2]), wherein damaged lysosomes generate tubules sorted into mobile vesicles. LYTL is orchestrated by the Parkinson's disease-associated kinase LRRK2 that recruits the motor adaptor protein and RHD family member JIP4 to lysosomes via phosphorylated RAB proteins. To identify new players involved in LYTL, we performed unbiased proteomics on isolated lysosomes after LRRK2 kinase inhibition. Our results demonstrate that there is recruitment of RILPL1 to ruptured lysosomes via LRRK2 activity to promote phosphorylation of RAB proteins at the lysosomal surface. RILPL1, which is also a member of the RHD family, enhances the clustering of LRRK2-positive lysosomes in the perinuclear area and causes retraction of LYTL tubules, in contrast to JIP4 which promotes LYTL tubule extension. Mechanistically, RILPL1 binds to p150Glued, a dynactin subunit, facilitating the transport of lysosomes and tubules to the minus end of microtubules. Further characterization of the tubulation process revealed that LYTL tubules move along tyrosinated microtubules, with tubulin tyrosination proving essential for tubule elongation. In summary, our findings emphasize the dynamic regulation of LYTL tubules by two distinct RHD proteins and pRAB effectors, serving as opposing motor adaptor proteins: JIP4, promoting tubulation via kinesin, and RILPL1, facilitating tubule retraction through dynein/dynactin. We infer that the two opposing processes generate a metastable lysosomal membrane deformation that facilitates dynamic tubulation events.

Phosphorylation of Rab29 at Ser185 regulates its localization and role in the lysosomal stress response in concert with LRRK2.

Rab proteins are small GTPases that regulate a myriad of intracellular membrane trafficking events. Rab29 is one of the Rab proteins phosphorylated by leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated kinase. Recent studies suggest that Rab29 regulates LRRK2, whereas the mechanism by which Rab29 is regulated remained unclear. Here, we report a novel phosphorylation in Rab29 that is not mediated by LRRK2 and occurs under lysosomal overload stress. Mass spectrometry analysis identified the phosphorylation site of Rab29 as Ser185, and cellular expression studies of phosphomimetic mutants of Rab29 at Ser185 unveiled the involvement of this phosphorylation in counteracting lysosomal enlargement. PKCα and PKCδ were deemed to be involved in this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKCs in the lysosomal stress response pathway comprised of Rab29 and LRRK2, and further underscore the importance of this pathway in the mechanisms underlying lysosomal homeostasis.

Preclinical modeling of chronic inhibition of the Parkinson\u2019s disease associated kinase LRRK2 reveals altered function of the endolysosomal system in vivo

The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson’s Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.

Acquired dysregulation of dopamine homeostasis reproduces features of Parkinson\u2019s disease

The catecholamine neurotransmitter dopamine has the potential to act as an endogenous neurotoxin when its vesicular sequestration is dysregulated. Despite postmortem analyses from patients with Parkinson’s disease that demonstrate decreased vesicular sequestration of dopamine with a corresponding increase in dopamine metabolism, dopamine’s contribution to nigrostriatal dopaminergic degeneration in Parkinson’s disease has been debated. Here, we present a new in vivo model demonstrating the induction of Parkinson’s disease-associated pathogenic mechanisms of degeneration resulting from acquired dysregulation of dopamine sequestration in nigrostriatal dopaminergic neurons in adult rats. Utilizing adeno-associated virus (serotype 2), viral-mediated small-hairpin RNA interference of endogenous vesicular monoamine transporter 2 (VMAT2) expression resulted in a loss of VMAT2 protein expression in transduced dopaminergic cell bodies in the substantia nigra with a corresponding loss of VMAT2 protein within the striatal terminals. The loss of VMAT2 resulted in an accumulation of cytosolic dopamine and subsequent increased dopamine metabolism, deficits in dopamine-mediated behaviors, and degeneration of nigrostriatal dopaminergic neurons that was rescued through reintroduction of exogenous VMAT2, demonstrating that the toxicity was specific to the loss of VMAT2. Analysis of parkinsonian pathogenic mechanisms of degeneration identified oxidative damage, activation of Parkinson’s disease-associated kinase LRRK2, and the formation of aberrant α-synuclein. This model demonstrates that a progressive acquired loss of VMAT2 expression in adulthood is sufficient to induce Parkinson’s disease-associated pathogenic mechanisms of degeneration and provides a new model to further investigate the consequences of cytosolic dopamine.

Interferon-\u03b3 signaling synergizes with LRRK2 in neurons and microglia derived from human induced pluripotent stem cells

Parkinson’s disease-associated kinase LRRK2 has been linked to IFN type II (IFN-γ) response in infections and to dopaminergic neuronal loss. However, whether and how LRRK2 synergizes with IFN-γ remains unclear. In this study, we employed dopaminergic neurons and microglia differentiated from patient-derived induced pluripotent stem cells carrying LRRK2 G2019S, the most common Parkinson’s disease-associated mutation. We show that IFN-γ enhances the LRRK2 G2019S-dependent negative regulation of AKT phosphorylation and NFAT activation, thereby increasing neuronal vulnerability to immune challenge. Mechanistically, LRRK2 G2019S suppresses NFAT translocation via calcium signaling and possibly through microtubule reorganization. In microglia, LRRK2 modulates cytokine production and the glycolytic switch in response to IFN-γ in an NFAT-independent manner. Activated LRRK2 G2019S microglia cause neurite shortening, indicating that LRRK2-driven immunological changes can be neurotoxic. We propose that synergistic LRRK2/IFN-γ activation serves as a potential link between inflammation and neurodegeneration in Parkinson’s disease.

A Cell-Based High-Throughput Screening Identified Two Compounds that Enhance PINK1-Parkin Signaling.

SummaryEarly-onset Parkinson's disease-associated PINK1-Parkin signaling maintains mitochondrial health. Therapeutic approaches for enhancing PINK1-Parkin signaling present a potential strategy for treating various diseases caused by mitochondrial dysfunction. We report two chemical enhancers of PINK1-Parkin signaling, identified using a robust cell-based high-throughput screening system. These small molecules, T0466 and T0467, activate Parkin mitochondrial translocation in dopaminergic neurons and myoblasts at low doses that do not induce mitochondrial accumulation of PINK1. Moreover, both compounds reduce unfolded mitochondrial protein levels, presumably through enhanced PINK1-Parkin signaling. These molecules also mitigate the locomotion defect, reduced ATP production, and disturbed mitochondrial Ca2+ response in the muscles along with the mitochondrial aggregation in dopaminergic neurons through reduced PINK1 activity in Drosophila. Our results suggested that T0466 and T0467 may hold promise as therapeutic reagents in Parkinson's disease and related disorders.

The crystal structure of pseudokinase PEAK1 (Sugen kinase 269) reveals an unusual catalytic cleft and a novel mode of kinase fold dimerization

The pseudokinase group encompasses some 10% of protein kinases, but pseudokinases diverge from canonical kinases in key motifs. The two members of the small new kinase family 3 (NKF3) group are considered pseudokinases. These proteins, pseudopodium-enriched atypical kinase 1 (PEAK1, Sugen kinase 269, or SgK269) and pragmin (Sugen kinase 223 or SgK223), act as scaffolds in growth factor signaling pathways, and both contain a kinase fold with degraded kinase motifs at their C termini. These kinases may harbor regions that mediate oligomerization or control other aspects of signal transduction, but a lack of structural information has precluded detailed investigations into their functional roles. In this study, we determined the X-ray crystal structure of the PEAK1 pseudokinase domain to 2.3 Å resolution. The structure revealed that the PEAK1 kinase-like domain contains a closed nucleotide-binding cleft that in this conformation may deleteriously affect nucleotide binding. Moreover, we found that N- and C-terminal extensions create a highly unusual all α-helical split-dimerization region, termed here the split helical dimerization (SHED) region. Sequence conservation analysis suggested that this region facilitates a dimerization mode that is conserved between PEAK1 and pragmin. Finally, we observed structural similarities between the PEAK1 SHED region and the C-terminal extension of the Parkinson's disease-associated kinase PINK1. In summary, PEAK1's kinase cleft is occluded, and its newly identified SHED region may promote an unexpected dimerization mode. Similarities of PEAK1 with the active kinase PINK1 may reclassify the latter as a member of the new kinase family 3 group.

Phos-tagなどのリン酸化プロテオミクス技術による疾患原因キナーゼの機能解析

Phos-tagなどのリン酸化プロテオミクス技術による疾患原因キナーゼの機能解析

'Up with the LRRK': a phosphorylated Rab10 assay for evaluation of LRRK2 activity and inhibitor engagement.

Protein kinases catalyse the addition of phosphate groups to Ser/Thr and Tyr residues in cognate substrates and are mutated or hyperactive in a variety of diseases, making them important targets for rationally designed drugs. A good example is the Parkinson's disease-associated kinase, leucine-rich repeat kinase 2 (LRRK2), which is mutated (and probably hyperactive) in a small, but significant, subset of patients. An exciting new approach for personalised therapy is the development of central nervous system (CNS)-active small-molecule kinase inhibitors, which could be employed to ‘normalise’ LRRK2 signalling in affected cell types. However, the development of such drugs requires validated assays for the analysis of target engagement and the assembly of a set of tools for interrogating LRRK2, and its substrates, both in vitro and in vivo. A new study published in the Biochemical Journal by Ito et al. establishes that a ‘Phos-tag’™-binding assay can be exploited to measure phosphorylation of a recently identified LRRK2 substrate (Ras-related protein in brain 10 (Rab10)), and to compare and contrast relative catalytic output from disease-associated LRRK2 mutants. Powerful in vivo chemical genetic approaches are also disclosed, in which the catalytic activity of LRRK2 is unequivocally linked to the extent of Rab10 phosphorylation and the effects of chemically distinct LRRK2 inhibitors are matched with on-target inhibition mechanisms mediated through LRRK2 and its substrate Rab10. These important findings should simplify the generic analysis of Rab10 phosphorylation in model biological systems and are likely to be applicable to other substrates of LRRK2 (or indeed other kinases) for which phospho-specific antibodies are either absent or unsatisfactory.

Abstract 3649: Broad profiling reveals opportunities for selective inhibition of disease-associated mutant kinases

Abstract Small molecule kinase inhibitors are promising therapeutic agents in a number of diseases, most notably cancer. However, mutations in kinases, both intrinsic and acquired, can drastically alter inhibitor sensitivity. To identify inhibitors of disease-associated mutant kinases, we conducted an unbiased functional screen of 182 small molecule kinase inhibitors against 76 mutated recombinant kinases arising from 21 cognate wild-type kinases. The results revealed novel lead compounds that were exquisitely selective for mutant kinases, including several that exhibited preferred inhibition of mutant kinases over their cognate wild-type kinases. This study provides a resource for the development of novel small molecule inhibitors against disease-associated mutant kinases and illustrates the potential of unbiased large-scale profiling as an approach to compound-centric kinase inhibitor discovery. Citation Format: Krisna C. Duong-Ly, Karthik Devarajan, Shuguang Liang, Kurumi Horiuchi, Yuren Wang, Haiching Ma, Jeffrey R. Peterson. Broad profiling reveals opportunities for selective inhibition of disease-associated mutant kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3649. doi:10.1158/1538-7445.AM2015-3649

A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation.

Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease-associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho-Ser65-ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.

Switching on ubiquitylation by phosphorylating a ubiquitous activator

The dysfunction of the E3 ubiquitin ligase Parkin is a key contributor to the development of early-onset Parkinson's disease. Parkin is responsible for the labelling of outer mitochondrial membrane proteins with the small modifier protein ubiquitin in response to oxidative stress. This ubiquitylation signals the clearance of the damaged mitochondria to preserve overall cell health. Recent structural and biochemical experiments have shown that native Parkin exists in an autoinhibited state that must be activated in order to unmask its full ubiquitylation potential. In a recent article in the Biochemical Journal (vol. 460, pp. 127-139), Kazlauskaite and co-workers identified that the Parkinson's disease-associated kinase PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1] can phosphorylate ubiquitin in response to mitochondrial depolarization. Furthermore, the authors demonstrated that phosphorylated ubiquitin can activate Parkin's E3 ligase activity and promote both increased autoubiquitylation and substrate ubiquitylation of the mitochondrial protein Miro1. The study provides exciting initial insights that show how PINK1 might activate ubiquitin through phosphorylation, and how this important regulatory step might switch on Parkin-mediated ubiquitylation.

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho−Ser65, suggesting that small molecules that mimic ubiquitinPhospho−Ser65 could hold promise as novel therapies for Parkinson's disease.

Pink1 Kinase and Its Membrane Potential (Δψ)-dependent Cleavage Product Both Localize to Outer Mitochondrial Membrane by Unique Targeting Mode

The Parkinson disease-associated kinase Pink1 is targeted to mitochondria where it is thought to regulate mitochondrial quality control by promoting the selective autophagic removal of dysfunctional mitochondria. Nevertheless, the targeting mode of Pink1 and its submitochondrial localization are still not conclusively resolved. The aim of this study was to dissect the mitochondrial import pathway of Pink1 by use of a highly sensitive in vitro assay. Mutational analysis of the Pink1 sequence revealed that its N terminus acts as a genuine matrix localization sequence that mediates the initial membrane potential (Δψ)-dependent targeting of the Pink1 precursor to the inner mitochondrial membrane, but it is dispensable for Pink1 import or processing. A hydrophobic segment downstream of the signal sequence impeded complete translocation of Pink1 across the mitochondrial inner membrane. Additionally, the C-terminal end of the protein promoted the retention of Pink1 at the outer membrane. Thus, multiple targeting signals featured by the Pink1 sequence result in the final localization of both the full-length protein and its major Δψ-dependent cleavage product to the cytosolic face of the outer mitochondrial membrane. Full-length Pink1 and deletion constructs resembling the natural Pink1 processing product were found to assemble into membrane potential-sensitive high molecular weight protein complexes at the mitochondrial surface and displayed similar cytoprotective effects when expressed in vivo, indicating that both species are functionally relevant.

A New Cytosolic Pathway from a Parkinson Disease-associated Kinase, BRPK/PINK1: ACTIVATION OF AKT VIA MTORC2

Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.

Kinetic Mechanistic Studies of Cdk5/p25-Catalyzed H1P Phosphorylation: Metal Effect and Solvent Kinetic Isotope Effect

Cdk5/p25 is a member of the cyclin-dependent, Ser/Thr kinase family and has been identified as one of the principle Alzheimer's disease-associated kinases that promote the formation of hyperphosphorylated tau, the major component of neurofibrillary tangles. We and others have been developing inhibitors of cdk5/p25 as possible therapeutic agents for Alzheimer's disease (AD). In support of these efforts, we examine the metal effect and solvent kinetic isotope effect on cdk5/p25-catalyzed H1P (a histone H-1-derived peptide) phosphorylation. Here, we report that a second Mg(2+) in addition to the one forming the MgATP complex is required to bind to cdk5/p25 for its catalytic activity. It activates cdk5/p25 by demonstrating an increase in k(cat) and induces a conformational change that favors ATP binding but has no effect on the binding affinity for the H1P peptide substrate. The binding of the second Mg(2+) does not change the binding order of substrates. The reaction follows the same rapid equilibrium random mechanism in the presence or absence of the second Mg(2+) as evidenced by initial velocity analysis and substrate analogue and product inhibition studies. A linear proton inventory with a normal SKIE of 2.0 +/- 0.1 in the presence of the second Mg(2+) was revealed and suggested a single proton transfer in the rate-limiting phosphoryl transfer step. The pH profile revealed a residue with a pK(a) of 6.5 that is most likely the general acid-base catalyst facilitating the proton transfer.

New biochemical approaches towards understanding the Parkinson's disease-associated kinase, LRRK2.

Some 5 years ago, it was first discovered that mutations in the gene encoding LRRK2 (leucine-rich repeat protein kinase 2) are tightly linked with a subset of familial PD (Parkinson's disease). Before this genetic association, LRRK2 had never been investigated biochemically. Now it is of utmost importance to establish whether LRRK2 is a bona fide kinase in vitro and in vivo and to understand how mutations of LRRK2 lead to the specific loss of dopaminergic neurons in the substantia nigra to cause PD. In spite of tremendous efforts in the research community, there is no consensus with regard to the magnitude of the enzymatic activity of LRRK2 mutant forms that segregate with PD owing, in part, to the lack of a highly sensitive kinase assay system, and it is still unclear whether an abnormal increase in kinase activity is responsible for LRRK2-associated PD. As described in this issue of the Biochemical Journal, Nichols et al.. have developed an extensive set of molecular tools, including an optimized peptide substrate for determining in vitro kinase activity of LRRK2, a set of kinase inhibitors that can be used to explore LRRK2 substrate specificity and biology, a much-needed murine-specific antibody for immunoprecipation, and efficient gene-silencing approaches. In the present commentary, we discuss some of the components of this new LRRK2 biochemical toolbox and how they can be used to better understand this enigmatic kinase.

PINK Participates in Parkinson’s Pathway

A neurodegenerative disease in mice with similarities to Parkinson’s disease in humans is caused by missense mutations in a serine protease known as HtrA2 (also called Omi). Plun-Favreau et al . describe a connection between this protein and PINK1, a mitochondrial protein kinase that is mutated in certain forms of Parkinson’s disease. HtrA2 functions to detect damaged or misfolded proteins caused by stress in the mitochondria. Misfolded proteins bind to a regulatory domain and activate the protease. The new work indicates that HtrA2 may also be regulated by phosphorylation in response to the stress-activated p38 MAP (mitogen-activated protein) kinase pathway. Although PINK1 binds to HtrA2 and was required for p38-activated phosphorylation and activation of HtrA2, PINK appeared not to directly phosphorylate HtrA2. Consistent with a role of this putative regulatory system in Parkinson’s disease, phosphorylation of HtrA2 was increased in samples of brain from patients with idiopathic Parkinson’s disease, as might be expected if mitochondrial stress in the diseased cells caused activation of the p38 pathway and consequent phosphorylation and activation of HtrA2. Commentary by Alnemri provides further context for the regulatory mechanism and its possible relevance to disease. H. Plun-Favreau, K. Klupsch, N. Moisoi, S. Gandhi, S. Kjaer, D. Frith, K. Harvey, E. Deas, R. J. Harvey, N. McDonald, N. W. Wood, L. M. Martins, J. Downward, The mitochondrial protease HtrA2 is regulated by Parkinson’s disease-associated kinase PINK1. Nat. Cell Biol. 9 , 1243-1252 (2007). [PubMed] E. S. Alnemri, HtrA2 and Parkinson’s disease: Think PINK? Nat. Cell Biol. 9 , 1227-1229 (2007). [PubMed]