The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinson’s disease patients, but little is known about its regulation and function. A common mutation changing Gly2019 to serine enhances catalytic activity, suggesting that small-molecule inhibitors might have utility in treating Parkinson’s disease. We employed various approaches to explore the substrate-specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower Km and nearly 2-fold higher Vmax than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating threonine over serine. We also observed that several ROCK (Rho kinase) inhibitors such as Y-27632 and H-1152, suppressed LRRK2 with similar potency to which they inhibited ROCK2. In contrast, GSK429286A, a selective ROCK inhibitor, did not significantly inhibit LRRK2. We also identified a mutant LRRK2[A2016T] that was normally active, but resistant to H-1152 and Y-27632, as well as sunitinib, a structurally unrelated multikinase inhibitor that, in contrast with other compounds, suppresses LRRK2, but not ROCK. We have also developed the first sensitive antibody that enables measurement of endogenous LRRK2 protein levels and kinase activity as well as shRNA (short hairpin RNA) methods to reduce LRRK2 expression. Finally, we describe a pharmacological approach to validate whether substrates are phosphorylated by LRRK2 and use this to provide evidence that LRRK2 may not be rate-limiting for the phosphorylation of the proposed substrate moesin. The findings of the present study will aid with the investigation of LRRK2.
leucine-rich repeat protein kinase-2 (LRRK2); moesin; Parkinson’s disease; phosphorylation; Rho kinase (ROCK)
Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type 11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.
movement disorder; neurodegeneration; protein kinase; signal transduction; tau tubulin kinase peptide substrate (TTBKtide)
The LKB1 tumor suppressor is a protein kinase that controls activity of adenine monophosphate-activated protein kinase (AMPK). LKB1 activity is regulated by the pseudokinase STRADα and the scaffolding protein MO25α, through an unknown, phosphorylation-independent, mechanism. We describe the structure of the core heterotrimeric LKB1-STRADα-MO25α complex, revealing an unusual allosteric mechanism of LKB1 activation. STRADα adopts a closed conformation typical of active protein kinases and binds LKB1 as a pseudosubstrate. STRADα and MO25α promote the active conformation of LKB1, which is stabilised by MO25α interacting with the LKB1 activation loop. This previously undescribed mechanism of kinase activation may be relevant to understanding the evolution of other pseudokinases. The structure also reveals how mutations found in Peutz-Jeghers syndrome and other cancers impair LKB1 function.
Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.
PINK1; Parkin; Parkinson's disease
Mutations in the WNK [with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser382). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl− co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser1261, in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser382 of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway.
coiled-coil domain; Na+/Cl− co-transporter (NCC); Na+/K+/2Cl− co-transporter 1 (NKCC1); osmotic stress; protein kinase; signal transduction; CATCHtide, cation chloride co-transporter peptide substrate; CCD, coiled-coil domain; CCT, conserved C-terminal; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; ES, embryonic stem; ERK, extracellular-signal-regulated kinase; GST, glutathione transferase; HEK-293, human embryonic kidney 293; LRRK2, leucine-repeat-rich kinase 2; MAPK, mitogen-activated protein kinase; NCC, Na+/Cl− co-transporter; NKCC, Na+/K+/2Cl− co-transporter; OSR, oxidative stress-responsive kinase; SPAK, SPS1-related proline/alanine-rich kinase; PI3K, phosphoinositide 3-kinase; WNK, with no lysine (K) kinase
Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro. We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated—namely Drosophila melanogaster (dPINK1), Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)—are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.
biochemistry; Parkinson's disease; kinase
The present study uses two independent genetic strategies to explore the requirement for Phosphoinositide dependent kinase-1 (PDK1) in the development of mature T cell populations from CD4/CD8 double positive thymocytes. The data show that CD4/CD8 double positive thymocytes that do not express PDK1 or express a catalytically inactive PDK1 mutant fail to produce mature invariant Vα14 NKT cells but can differentiate to conventional CD4, CD8 or regulatory T cell subsets in the thymus. The PDK1 requirement for Vα14 NKT cell development reflects that these cells require the PDK1 substrate Protein Kinase B (PKB, also called Akt) to meet the metabolic demands for proliferative expansion in response to Interleukin 15 or antigen receptor stimulation. There is also constitutive PDK1 signaling in conventional α/β T cells that is not required for lineage commitment of these cells but fine-tunes the expression of coreceptors and adhesion molecules. Also, while PDK1 is dispensable for thymic development of conventional α/β T cells, peripheral cells are substantially reduced. This reflects a PDK1 requirement for lymphopenia-induced proliferation, a process necessary for initial population of the peripheral T cell niche in neonatal mice. PDK1 is thus indispensable for T cell developmental programs but the timing of the PDK1 requirement is unique to different T cell subpopulations.
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.
cytoplasmic localization; 14-3-3 protein; leucine-rich repeat protein kinase 2 (LRRK2); Parkinson's disease; pathogenic mutation; phosphorylation; CDC, cell division cycle; DIG, digoxigenin; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; FBS, fetal bovine serum; GFP, green fluorescent protein; HEK-293, human embryonic kidney; Hsp90, heat-shock protein 90; IPI, International Protein Index; KLH, keyhole-limpet haemocyanin; LRRK2, leucine-rich repeat protein kinase 2; MARK3, microtubule affinity-regulating kinase 3; PD, Parkinson's disease; ROC, Ras of complex GTPase domain; COR, C-terminal of ROC; SILAC, stable isotope labelling of amino acids; TBST, Tris-buffered saline with Tween 20
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's
disease patients. Since a common mutation that replaces Gly2019 with a serine residue
enhances kinase catalytic activity, small-molecule LRRK2 inhibitors might have utility in treating
Parkinson's disease. However, the effectiveness of inhibitors is difficult to assess, as no
physiological substrates or downstream effectors have been identified that could be exploited to
develop a robust cell-based assay. We recently established that LRRK2 bound 14-3-3 protein isoforms
via its phosphorylation of Ser910 and Ser935. In the present study we show
that treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson's
disease patient harbouring a homozygous LRRK2(G2019S) mutation with two structurally unrelated
inhibitors of LRRK2 (H-1152 or sunitinib) induced dephosphorylation of endogenous LRRK2 at
Ser910 and Ser935, thereby disrupting 14-3-3 interaction. Our results suggest
that H-1152 and sunitinib induce dephosphorylation of Ser910 and Ser935 by
inhibiting LRRK2 kinase activity, as these compounds failed to induce significant dephosphorylation
of a drug-resistant LRRK2(A2016T) mutant. Moreover, consistent with the finding that
non-14-3-3-binding mutants of LRRK2 accumulated within discrete cytoplasmic pools resembling
inclusion bodies, we observed that H-1152 causes LRRK2 to accumulate within inclusion bodies. These
findings indicate that dephosphorylation of Ser910/Ser935, disruption of
14-3-3 binding and/or monitoring LRRK2 cytoplasmic localization can be used as an assay to assess
the relative activity of LRRK2 inhibitors in vivo. These results will aid the
elaboration and evaluation of LRRK2 inhibitors. They will also stimulate further research to
understand how phosphorylation of Ser910 and Ser935 is controlled by LRRK2,
and establish any relationship to development of Parkinson's disease.
cell-based assay; drug discovery; 14-3-3 protein kinase inhibitor; leucine-rich repeat protein kinase 2 (LRRK2); Parkinson's disease; protein phosphorylation; DIG, digoxigenin; DMEM, Dulbecco's modified Eagle's medium; EBV, Epstein–Barr virus; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; GFP, green fluorescent protein; HEK, human embryonic kidney; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; KLH, keyhole-limpet haemocyanin; LRRK2, leucine-rich repeat protein kinase 2; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; mTOR, mammalian target of rapamycin; MYPT, myosin phosphatase-targeting; NP-40, Nonidet P40; PFA, paraformaldehyde; PI3K, phosphoinositide 3-kinase; ROCK, Rho-kinase; TBST, Tris-buffered saline with Tween 20
The conformation of the pseudokinase STRADα, which is regulated by binding to ATP and to the scaffolding protein MO25α, is key to the activiation of the LKB1 tumor suppressor complex.
Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADα in complex with MO25α. The structure reveals an intricate web of interactions between STRADα and MO25α involving the αC-helix of STRADα, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADα binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADα for MO25α, and conversely, binding of MO25α promotes interaction of STRADα with ATP. Mutagenesis studies reveal that association of STRADα with either ATP or MO25α is essential for LKB1 activation. We conclude that ATP and MO25α cooperate to maintain STRADα in an “active” closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADα that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE) syndrome. We demonstrate this mutation destabilizes STRADα and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADα to activate LKB1 is dependent on a closed “active” conformation, aided by ATP and MO25α binding. Thus, the function of STRADα is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.
There are 518 human protein kinases that are responsible for orchestrating the phosphorylation-dependant signal transduction events that regulate almost all cellular processes. Curiously, approximately 10% of protein kinases lack one or more catalytic residues, and these kinases have been termed pseudokinases. It has been proposed that some pseudokinases act as scaffolds, bringing together proteins involved in signalling networks. Here, we report the structure of the pseudokinase STRADα in complex with the adaptor protein MO25α; together these two proteins regulate the LKB1 tumour suppressor kinase. Despite lacking several key catalytic residues, STRADα binds ATP and adopts an active conformation typical of catalytically competent kinases. The affinity of STRADα for ATP is enhanced by MO25α and vice versa. We go on to demonstrate through mutagenesis studies that binding to both ATP and MO25α is essential for the activation of LKB1. Our data suggest that STRADα exerts its functions through an active conformation, not through actual catalytic activity, thus raising the possibility that pseudokinases regulate signalling networks by adopting different structural conformations.
Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine–rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane–coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.
We have characterized Drosophila melanogaster ACK (DACK), one of two members of the ACK family of nonreceptor tyrosine kinases in Drosophila. The ACKs are likely effectors for the small GTPase Cdc42, but signaling by these proteins remains poorly defined. ACK family tyrosine kinase activity functions downstream of Drosophila Cdc42 during dorsal closure of the embryo, as overexpression of DACK can rescue the dorsal closure defects caused by dominant-negative Dcdc42. Similar to known participants in dorsal closure, DACK is enriched in the leading edge cells of the advancing epidermis, but it does not signal through activation of the Jun amino-terminal kinase cascade operating in these cells. Transcription of DACK is responsive to changes in Dcdc42 signaling specifically at the leading edge and in the amnioserosa, two tissues involved in dorsal closure. Unlike other members of the ACK family, DACK does not contain a conserved Cdc42-binding motif, and transcriptional regulation may be one route by which Dcdc42 can affect DACK function. Expression of wild-type and kinase-dead DACK transgenes in embryos, and in the developing wing and eye, reveals that ACK family tyrosine kinase activity is involved in a range of developmental events similar to that of Dcdc42.