LRRK1 functions as a scaffold for PTP1B-mediated EGFR sorting into ILVs at the ER-endosome contact site.

Proper control of epidermal growth factor receptor (EGFR) signaling is important for maintaining cellular homeostasis. Given that EGFR signaling occurs at the plasma membrane and endosomes following internalization, endosomal trafficking of EGFR spatiotemporally regulates EGFR signaling. In this process, leucine-rich repeat kinase 1 (LRRK1) has multiple roles in kinase activity-dependent transport of EGFR-containing endosomes and kinase-independent sorting of EGFR into the intraluminal vesicles (ILVs) of multivesicular bodies. Active, phosphorylated EGFR inactivates the LRRK1 kinase activity by phosphorylating Y944. In this study, we demonstrate that LRRK1 facilitates EGFR dephosphorylation by PTP1B (also known as PTPN1), an endoplasmic reticulum (ER)-localized protein tyrosine phosphatase, at the ER-endosome contact site, after which EGFR is sorted into the ILVs of endosomes. LRRK1 is required for the PTP1B-EGFR interaction in response to EGF stimulation, resulting in the downregulation of EGFR signaling. Furthermore, PTP1B activates LRRK1 by dephosphorylating pY944 on the contact site, which promotes the transport of EGFR-containing endosomes to the perinuclear region. These findings provide evidence that the ER-endosome contact site functions as a hub for LRRK1-dependent signaling that regulates EGFR trafficking.

The ULK complex-LRRK1 axis regulates Parkin-mediated mitophagy via Rab7 Ser-72 phosphorylation.

Mitophagy, a type of selective autophagy, specifically targets damaged mitochondria. The ULK complex regulates Parkin-mediated mitophagy, but the mechanism through which the ULK complex initiates mitophagosome formation remains unknown. The Rab7 GTPase (herein referring to Rab7a) is a key initiator of mitophagosome formation, and Ser-72 phosphorylation of Rab7 is important for this process. We have previously identified LRRK1 as a protein kinase responsible for Rab7 Ser-72 phosphorylation. In this study, we investigated the role of LRRK1 in mitophagy. We showed that LRRK1 functions downstream of ULK1 and ULK2 in Parkin-mediated mitophagy. Furthermore, we demonstrated that ectopic targeting of active LRRK1 to mitochondria is sufficient to induce the Ser-72 phosphorylation of Rab7, circumventing the requirement for ATG13, a component of the ULK complex. Thus, the ULK complex recruits LRRK1 to mitochondria by interacting with ATG13 to initiate mitophagosome formation. This study highlights the crucial role of the ULK complex-LRRK1 axis in the regulation of Parkin-mediated mitophagy.

LRRK1-mediated NDEL1 phosphorylation promotes cilia disassembly via dynein-2-driven retrograde intraflagellar transport.

Primary cilia are antenna-like organelles that regulate growth and development via extracellular signals. However, the molecular mechanisms underlying cilia dynamics, particularly those regulating their disassembly, are not well understood. Here, we show that leucine-rich repeat kinase 1 (LRRK1) plays a role in regulating cilia disassembly. The depletion of LRRK1 impairs primary cilia resorption following serum stimulation in cultured cells. Polo-like kinase 1 (PLK1) plays an important role in this process. During ciliary resorption, PLK1 phosphorylates LRRK1 at the primary cilia base, resulting in its activation. We identified nuclear distribution protein nudE-like 1 (NDEL1), which is known to positively regulate cilia disassembly, as a target of LRRK1 phosphorylation. Whereas LRRK1 phosphorylation of NDEL1 on Ser-155 promotes NDEL1 interaction with the intermediate chains of cytoplasmic dynein-2, it is also crucial for triggering ciliary resorption through dynein-2-driven retrograde intraflagellar transport. These findings provide evidence that a novel PLK1-LRRK1-NDEL1 pathway regulates cilia disassembly.

Histidine dephosphorylation of the Gß protein GPB-1 promotes axon regeneration in C. elegans.

Histidine phosphorylation is an emerging noncanonical protein phosphorylation in animals, yet its physiological role remains largely unexplored. The protein histidine phosphatase (PHPT1) was recently identified for the first time in mammals. Here, we report that PHIP-1, an ortholog of PHPT1 in Caenorhabditis elegans, promotes axon regeneration by dephosphorylating GPB-1 Gß at His-266 and inactivating GOA-1 Goα signaling, a negative regulator of axon regeneration. Overexpression of the histidine kinase NDK-1 also inhibits axon regeneration via GPB-1 His-266 phosphorylation. Thus, His-phosphorylation plays an antiregenerative role in C. elegans. Furthermore, we identify a conserved UNC-51/ULK kinase that functions in autophagy as a PHIP-1-binding protein. We demonstrate that UNC-51 phosphorylates PHIP-1 at Ser-112 and activates its catalytic activity and that this phosphorylation is required for PHIP-1-mediated axon regeneration. This study reveals a molecular link from ULK to protein histidine phosphatase, which facilitates axon regeneration by inhibiting trimeric G protein signaling.

CDK14 Promotes Axon Regeneration by Regulating the Noncanonical Wnt Signaling Pathway in a Kinase-Independent Manner.

The postinjury regenerative capacity of neurons is known to be mediated by a complex interaction of intrinsic regenerative pathways and external cues. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the nonmuscle myosin light chain-4 (MLC-4) phosphorylation signaling pathway. In this study, we have identified svh-16/cdk-14, a mammalian CDK14 homolog, as a positive regulator of axon regeneration in motor neurons. We then isolated the CDK-14-binding protein MIG-5/Disheveled (Dsh) and found that EGL-20/Wnt and the MIG-1/Frizzled receptor (Fz) are required for efficient axon regeneration. Further, we demonstrate that CDK-14 activates EPHX-1, the C. elegans homolog of the mammalian ephexin Rho-type GTPase guanine nucleotide exchange factor (GEF), in a kinase-independent manner. EPHX-1 functions as a GEF for the CDC-42 GTPase, inhibiting myosin phosphatase, which maintains MLC-4 phosphorylation. These results suggest that CDK14 activates the RhoGEF-CDC42-MLC phosphorylation axis in a noncanonical Wnt signaling pathway that promotes axon regeneration.SIGNIFICANCE STATEMENT Noncanonical Wnt signaling is mediated by Frizzled receptor (Fz), Disheveled (Dsh), Rho-type GTPase, and nonmuscle myosin light chain (MLC) phosphorylation. This study identified svh-16/cdk-14, which encodes a mammalian CDK14 homolog, as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that CDK-14 binds to MIG-5/Dsh, and that EGL-20/Wnt, MIG-1/Fz, and EPHX-1/RhoGEF are required for axon regeneration. The phosphorylation-mimetic MLC-4 suppressed axon regeneration defects in mig-1, cdk-14, and ephx-1 mutants. CDK-14 mediates kinase-independent activation of EPHX-1, which functions as a guanine nucleotide exchange factor for CDC-42 GTPase. Activated CDC-42 inactivates myosin phosphatase and thereby maintains MLC phosphorylation. Thus, the noncanonical Wnt signaling pathway controls axon regeneration via the CDK-14-EPHX-1-CDC-42-MLC phosphorylation axis.

The Integrin Signaling Network Promotes Axon Regeneration via the Src-Ephexin-RhoA GTPase Signaling Axis.

Axon regeneration is an evolutionarily conserved process essential for restoring the function of damaged neurons. In Caenorhabditis elegans hermaphrodites, initiation of axon regeneration is regulated by the RhoA GTPase-ROCK (Rho-associated coiled-coil kinase)-regulatory nonmuscle myosin light-chain phosphorylation signaling pathway. However, the upstream mechanism that activates the RhoA pathway remains unknown. Here, we show that axon injury activates TLN-1/talin via the cAMP-Epac (exchange protein directly activated by cAMP)-Rap GTPase cascade and that TLN-1 induces multiple downstream events, one of which is integrin inside-out activation, leading to the activation of the RhoA-ROCK signaling pathway. We found that the nonreceptor tyrosine kinase Src, a key mediator of integrin signaling, activates the Rho guanine nucleotide exchange factor EPHX-1/ephexin by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the C. elegans integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.SIGNIFICANCE STATEMENT The ability of axons to regenerate after injury is governed by cell-intrinsic regeneration pathways. We have previously demonstrated that the Caenorhabditis elegans RhoA GTPase-ROCK (Rho-associated coiled-coil kinase) pathway promotes axon regeneration by inducing MLC-4 phosphorylation. In this study, we found that axon injury activates TLN-1/talin through the cAMP-Epac (exchange protein directly activated by cAMP)-Rap GTPase cascade, leading to integrin inside-out activation, which promotes axonal regeneration by activating the RhoA signaling pathway. In this pathway, SRC-1/Src acts downstream of integrin activation and subsequently activates EPHX-1/ephexin RhoGEF by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the C. elegans integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.

BRCA1-BARD1 Regulates Axon Regeneration in Concert with the Gqα-DAG Signaling Network.

The breast cancer susceptibility protein BRCA1 and its partner BRCA1-associated RING domain protein 1 (BARD1) form an E3-ubiquitin (Ub) ligase complex that acts as a tumor suppressor in mitotic cells. However, the roles of BRCA1-BARD1 in postmitotic cells, such as neurons, remain poorly defined. Here, we report that BRC-1 and BRD-1, the Caenorhabditis elegans orthologs of BRCA1 and BARD1, are required for adult-specific axon regeneration, which is positively regulated by the EGL-30 Gqα-diacylglycerol (DAG) signaling pathway. This pathway is downregulated by DAG kinase (DGK), which converts DAG to phosphatidic acid (PA). We demonstrate that inactivation of DGK-3 suppresses the brc-1 brd-1 defect in axon regeneration, suggesting that BRC-1-BRD-1 inhibits DGK-3 function. Indeed, we show that BRC-1-BRD-1 poly-ubiquitylates DGK-3 in a manner dependent on its E3 ligase activity, causing DGK-3 degradation. Furthermore, we find that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. These results suggest that the BRC-1-BRD-1 complex regulates axon regeneration in concert with the Gqα-DAG signaling network. Thus, this study describes a new role for breast cancer proteins in fully differentiated neurons and the molecular mechanism underlying the regulation of axon regeneration in response to nerve injury.SIGNIFICANCE STATEMENT BRCA1-BRCA1-associated RING domain protein 1 (BARD1) is an E3-ubiquitin (Ub) ligase complex acting as a tumor suppressor in mitotic cells. The roles of BRCA1-BARD1 in postmitotic cells, such as neurons, remain poorly defined. We show here that Caenorhabditis elegans BRC-1/BRCA1 and BRD-1/BARD1 are required for adult-specific axon regeneration, a process that requires high diacylglycerol (DAG) levels in injured neurons. The DAG kinase (DGK)-3 inhibits axon regeneration by reducing DAG levels. We find that BRC-1-BRD-1 poly-ubiquitylates and degrades DGK-3, thereby keeping DAG levels elevated and promoting axon regeneration. Furthermore, we demonstrate that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. Thus, this study describes a new role for BRCA1-BARD1 in fully-differentiated neurons.

Caenorhabditis elegans F-Box Protein Promotes Axon Regeneration by Inducing Degradation of the Mad Transcription Factor.

In Caenorhabditis elegans, axon regeneration is activated by a signaling cascade through the receptor tyrosine kinase (RTK) SVH-2. Axonal injury induces svh-2 gene expression by degradation of the Mad-like transcription factor MDL-1. In this study, we identify the svh-24/sdz-33 gene encoding a protein containing F-box and F-box-associated domains as a regulator of axon regeneration in motor neurons. We find that sdz-33 is required for axon injury-induced svh-2 expression. SDZ-33 targets MDL-1 for poly-ubiquitylation and degradation. Furthermore, we demonstrate that SDZ-33 promotes axotomy-induced nuclear degradation of MDL-1, resulting in the activation of svh-2 expression in animals. These results suggest that the F-box protein is required for RTK signaling in the control of axon regeneration.SIGNIFICANCE STATEMENT In Caenorhabditis elegans, axon regeneration is positively regulated by the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury via the Ets-like transcription factor ETS-4, whose transcriptional activity is inhibited by the Mad-like transcription factor MDL-1. Axon injury leads to the degradation of MDL-1, and this is linked to the activation of ETS-4 transcriptional activity. In this study, we identify the sdz-33 gene encoding a protein containing an F-box domain as a regulator of axon regeneration. We demonstrate that MDL-1 is poly-ubiquitylated and degraded through the SDZ-33-mediated 26S proteasome pathway. These results reveal that an F-box protein promotes axon regeneration by degrading the Mad transcription factor.

TDP2 negatively regulates axon regeneration by inducing SUMOylation of an Ets transcription factor.

In Caenorhabditis elegans, the JNK MAP kinase (MAPK) pathway is important for axon regeneration. The JNK pathway is activated by a signaling cascade consisting of the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury in a process involving the transcription factors ETS-4 and CEBP-1. Here, we find that svh-14/mxl-1, a gene encoding a Max-like transcription factor, is required for activation of svh-2 expression in response to axonal injury. We show that MXL-1 binds to and inhibits the function of TDPT-1, a C. elegans homolog of mammalian tyrosyl-DNA phosphodiesterase 2 [TDP2; also called Ets1-associated protein II (EAPII)]. Deletion of tdpt-1 suppresses the mxl-1 defect, but not the ets-4 defect, in axon regeneration. TDPT-1 induces SUMOylation of ETS-4, which inhibits ETS-4 transcriptional activity, and MXL-1 counteracts this effect. Thus, TDPT-1 interacts with two different transcription factors in axon regeneration.

C. elegans Tensin Promotes Axon Regeneration by Linking the Met-like SVH-2 and Integrin Signaling Pathways.

Axon regeneration is a conserved mechanism induced by axon injury that initiates a neuronal response leading to regrowth of the axon. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the JNK MAP kinase (MAPK) pathway. We have previously identified a number of genes affecting the JNK pathway using an RNAi-based screen. Analysis of these genes, called the svh genes, has shed new light on the regulation of axon regeneration, revealing the involvement of a signaling cascade consisting of a growth factor SVH-1 and its receptor, the tyrosine kinase SVH-2. Here, we characterize the svh-6/tns-1 gene, which is a homolog of mammalian tensin, and show that it is a positive regulator of axon regeneration in motor neurons. We demonstrate that TNS-1 interacts with tyrosine-autophosphorylated SVH-2 and the integrin ß subunit PAT-3 via its SH2 and PTB domains, respectively, to promote axon regeneration. These results suggest that TNS-1 acts as an adaptor to link the SVH-2 and integrin signaling pathways.SIGNIFICANCE STATEMENT The Caenorhabditis elegans JNK MAPK pathway regulates the initiation of axon regeneration. Previously, we showed that a signaling cascade consisting of the HGF-like growth factor SVH-1 and its Met-like receptor tyrosine kinase SVH-2 promotes axon regeneration through activation of the JNK pathway. In this study, we show that the C. elegans tensin, TNS-1, is required for efficient regeneration after axon injury. Phosphorylation of SVH-2 on tyrosine mediates its interaction with the SH2 domain of TNS-1 to positively regulate axon regeneration. Furthermore, TNS-1 interacts via its PTB domain with the integrin ß subunit PAT-3. These results suggest that TNS-1 plays a critical role in the regulation of axon regeneration by linking the SVH-2 and integrin signaling pathways.

LRRK1 phosphorylation of Rab7 at S72 links trafficking of EGFR-containing endosomes to its effector RILP.

Ligand-induced activation of epidermal growth factor receptor (EGFR) initiates trafficking events that re-localize the receptor from the cell surface to intracellular endocytic compartments. EGFR-containing endosomes are transported to lysosomes for degradation by the dynein-dynactin motor protein complex. However, this cargo-dependent endosomal trafficking mechanism remains largely uncharacterized. Here, we show that GTP-bound Rab7 is phosphorylated on S72 by leucine-rich repeat kinase 1 (LRRK1) at the endosomal membrane. This phosphorylation promotes the interaction of Rab7 (herein referring to Rab7a) with its effector RILP, resulting in recruitment of the dynein-dynactin complex to Rab7-positive vesicles. This, in turn, facilitates the dynein-driven transport of EGFR-containing endosomes toward the perinuclear region. These findings reveal a mechanism regulating the cargo-specific trafficking of endosomes.

The C. elegans BRCA2-ALP/Enigma Complex Regulates Axon Regeneration via a Rho GTPase-ROCK-MLC Phosphorylation Pathway.

The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the mechanisms regulating axon regeneration are not well understood. Here, we identify the brc-2 gene encoding a homolog of the mammalian BRCA2 tumor suppressor as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that the RHO-1/Rho GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC-4/MLC) phosphorylation signaling pathway regulates axon regeneration. BRC-2 functions between RHO-1 and LET-502, suggesting that BRC-2 is required for the activation of LET-502 by RHO-1-GTP. We also find that one component that interacts with BRC-2, the ALP (α-actinin-associated LIM protein)/Enigma protein ALP-1, is required for regeneration and acts between LET-502 and MLC-4 phosphorylation. Furthermore, we demonstrate that ALP-1 associates with LET-502 and MLC-4. Thus, ALP-1 serves as a platform to activate MLC-4 phosphorylation mediated by the RHO-1-LET-502 signaling pathway.

Phosphatidylserine exposure mediated by ABC transporter activates the integrin signaling pathway promoting axon regeneration.

Following axon injury, a cascade of signaling events is triggered to initiate axon regeneration. However, the mechanisms regulating axon regeneration are not well understood at present. In Caenorhabditis elegans, axon regeneration utilizes many of the components involved in phagocytosis, including integrin and Rac GTPase. Here, we identify the transthyretin (TTR)-like protein TTR-11 as a component functioning in axon regeneration upstream of integrin. We show that TTR-11 binds to both the extracellular domain of integrin-α and phosphatidylserine (PS). Axon injury induces the accumulation of PS around the injured axons in a manner dependent on TTR-11, the ABC transporter CED-7, and the caspase CED-3. Furthermore, we demonstrate that CED-3 activates CED-7 during axon regeneration. Thus, TTR-11 functions to link the PS injury signal to activation of the integrin pathway, which then initiates axon regeneration.

TAK1 determines susceptibility to endoplasmic reticulum stress and leptin resistance in the hypothalamus.

Sustained endoplasmic reticulum (ER) stress disrupts normal cellular homeostasis and leads to the development of many types of human diseases, including metabolic disorders. TAK1 (also known as MAP3K7) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family and is activated by a diverse set of inflammatory stimuli. Here, we demonstrate that TAK1 regulates ER stress and metabolic signaling through modulation of lipid biogenesis. We found that deletion of Tak1 increased ER volume and facilitated ER-stress tolerance in cultured cells, which was mediated by upregulation of sterol-regulatory-element-binding protein (SREBP)-dependent lipogenesis. In the in vivo setting, central nervous system (CNS)-specific Tak1 deletion upregulated SREBP-target lipogenic genes and blocked ER stress in the hypothalamus. Furthermore, CNS-specific Tak1 deletion prevented ER-stress-induced hypothalamic leptin resistance and hyperphagic obesity under a high-fat diet (HFD). Thus, TAK1 is a crucial regulator of ER stress in vivo, which could be a target for alleviation of ER stress and its associated disease conditions.

Chaperone complex BAG2-HSC70 regulates localization of Caenorhabditis elegans leucine-rich repeat kinase LRK-1 to the Golgi.

Mutations in LRRK2 are linked to autosomal dominant forms of Parkinson's disease. We identified two human proteins that bind to LRRK2: BAG2 and HSC70, which are known to form a chaperone complex. We characterized the role of their Caenorhabditis elegans homologues, UNC-23 and HSP-1, in the regulation of LRK-1, the sole homologue of human LRRK2. In C. elegans, LRK-1 determines the polarized sorting of synaptic vesicle (SV) proteins to the axons by excluding SV proteins from the dendrite-specific transport machinery in the Golgi. In unc-23 mutants, SV proteins are localized to both presynaptic and dendritic endings in neurons, a phenotype also observed in lrk-1 deletion mutants. Furthermore, we isolated mutations in the hsp-1 gene that can suppress the unc-23, but not the lrk-1 defect. We show that UNC-23 determines LRK-1 localization to the Golgi apparatus in cooperation with HSP-1. These results describe a chaperone-dependent mechanism through which LRK-1 localization is regulated.

PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2.

Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation.

LRRK1-phosphorylated CLIP-170 regulates EGFR trafficking by recruiting p150Glued to microtubule plus ends.

The binding of ligand to epidermal growth factor receptor (EGFR) causes the receptor to become activated and stimulates the endocytosis of EGFR. Early endosomes containing activated EGFR migrate along microtubules as they mature into late endosomes. We have recently shown that LRRK1, which is related to the familial Parkinsonism gene product Park8 (also known as LRRK2), regulates this EGFR transport in a manner dependent on LRRK1 kinase activity. However, the downstream targets of LRRK1 that might modulate this transport function have not been identified. Here, we identify CLIP-170 (also known as CLIP1), a microtubule plus-end protein, as a substrate of LRRK1. LRRK1 phosphorylates CLIP-170 at Thr1384, located in its C-terminal zinc knuckle motif, and this promotes the association of CLIP-170 with dynein-dynactin complexes. We find that LRRK1-mediated phosphorylation of CLIP-170 causes the accumulation of p150(Glued) (also known as DCTN1) a subunit of dynactin, at microtubule plus ends, thereby facilitating the migration of EGFR-containing endosomes. Thus, our findings provide new mechanistic insights into the dynein-driven transport of EGFR.