mRNA location and translation rate determine protein targeting to dual destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.

A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2.

Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously in vitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion.

RNA in situ conformation sequencing reveals novel long-range RNA structures with impact on splicing.

Over recent years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. An increasing number of human disorders are now being associated with splicing defects; hence it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is a method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose pairs of conserved complementary regions (PCCRs) that were predicted in silico with the results of RIC-seq experiments conducted in seven human cell lines. We show statistically that RIC-seq support of PCCRs correlates with their properties, such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Exons enclosed in PCCRs that are supported by RIC-seq tend to have weaker splice sites and lower inclusion rates, which is indicative of post-transcriptional splicing regulation mediated by RNA structure. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show, using antisense nucleotides and minigene mutagenesis, that PCCRs in two disease-associated human genes, PHF20L1 and CASK, and also PCCRs in their murine orthologs, impact alternative splicing. In sum, we demonstrate how RIC-seq experiments can be used to discover functional long-range RNA structures, and particularly those that regulate alternative splicing.

Golgi localization of the LIN-2/7/10 complex points to a role in basolateral secretion of LET-23 EGFR in the Caenorhabditiselegans vulval precursor cells.

The evolutionarily conserved LIN-2 (CASK)/LIN-7 (Lin7A-C)/LIN-10 (APBA1) complex plays an important role in regulating spatial organization of membrane proteins and signaling components. In Caenorhabditiselegans, the complex is essential for the development of the vulva by promoting the localization of the sole Epidermal growth factor receptor (EGFR) ortholog LET-23 to the basolateral membrane of the vulva precursor cells where it can specify the vulval cell fate. To understand how the LIN-2/7/10 complex regulates receptor localization, we determined its expression and localization during vulva development. We found that LIN-7 colocalizes with LET-23 EGFR at the basolateral membrane, whereas the LIN-2/7/10 complex colocalizes with LET-23 EGFR at cytoplasmic punctae that mostly overlap with the Golgi. Furthermore, LIN-10 recruits LIN-2, which in turn recruits LIN-7. We demonstrate that the complex forms in vivo with a particularly strong interaction and colocalization between LIN-2 and LIN-7, consistent with them forming a subcomplex. Thus, the LIN-2/7/10 complex forms on the Golgi on which it likely targets LET-23 EGFR trafficking to the basolateral membrane rather than functioning as a tether.

CASK pathogenic variant which expands the clinical spectrum for MICPCH syndrome in an adult patient.

The CASK gene and its product protein kinase have been associated with microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome and various other neurodevelopmental disorders. Clinical presentation is highly variable and generally includes intellectual disability, neurological disorders, and dysmorphic features, at a minimum. We present the case of one of the oldest known currently living patients with MICPCH syndrome with additional features not previously described in the literature (midface retrusion, macroglossia, dental crowding, adolescent-onset contractures at large joints, laxity at finger joints, and prominent wrist dystonia). Progressive hypertonicity throughout the patient's life has been managed with serial botulinum toxin injections. A comprehensive multimodal care team including physiatry, physical therapy, exercise therapy, and audiology has been assisting her with hearing deficits, communication skills, and mobility. This potentially expands the phenotype of MICPCH syndrome and provides information about the management of this condition into adulthood.

A Threshold Helium Leakage Detection Switch with Ultra Low Power Operation.

Detecting helium leakage is important in many applications, such as in dry cask nuclear waste storage systems. This work develops a helium detection system based on the relative permittivity (dielectric constant) difference between air and helium. This difference changes the status of an electrostatic microelectromechanical system (MEMS) switch. The switch is a capacitive-based device and requires a very negligible amount of power. Exciting the switch's electrical resonance enhances the MEMS switch sensitivity to detect low helium concentration. This work simulates two different MEMS switch configurations: a cantilever-based MEMS modeled as a single-degree-freedom model and a clamped-clamped beam MEMS molded using the COMSOL Multiphysics finite-element software. While both configurations demonstrate the switch's simple operation concept, the clamped-clamped beam was selected for detailed parametric characterization due to its comprehensive modeling approach. The beam detects at least 5% helium concentration levels when excited at 3.8 MHz, near electrical resonance. The switch performance decreases at lower excitation frequencies or increases the circuit resistance. The MEMS sensor detection level was relatively immune to beam thickness and parasitic capacitance changes. However, higher parasitic capacitance increases the switch's susceptibility to errors, fluctuations, and uncertainties.

Pathogenic variants in CASK: Expanding the genotype-phenotype correlations.

Pathogenic variants in CASK, an X-linked gene that plays a role in brain development and synaptic function, are the cause of both microcephaly with pontine and cerebellar hypoplasia (MICPCH), and X-linked intellectual disability (XLID) with or without nystagmus. MICPCH is caused by loss of function variants in CASK, typically affects females, and is associated with moderate-to-severe intellectual disability (ID). Additional findings, present in about one-third of individuals, include feeding difficulties, ophthalmologic issues, hypertonicity, epilepsy, and sensorineural hearing loss. Only a few affected males with MICPCH phenotype have been reported and most have had profound developmental disability and intractable epilepsy. The XLID phenotype is typically caused by missense variants and most often manifests in males; carrier females are mildly affected or unaffected. Nystagmus is often present. In total, over 175 patients have been reported in the literature. We now report an additional 11 patients with pathogenic variants in CASK that expand these phenotypes and reported genotype-phenotype correlations.

Reciprocal Xp11.4p11.3 microdeletion/microduplication spanning USP9X, DDX3X, and CASK genes in two patients with syndromic intellectual disability.

Only a few patients with deletions or duplications at Xp11.4, bridging USP9X, DDX3X, and CASK genes, have been described so far. Here, we report on a female harboring a de novo Xp11.4p11.3 deletion and a male with an overlapping duplication inherited from an unaffected mother, presenting with syndromic intellectual disability. We discuss the role of USP9X, DDX3X, and CASK genes in human development and describe the effects of Xp11.4 deletion and duplications in female and male patients, respectively.

Missense mutations in CASK, coding for the calcium-/calmodulin-dependent serine protein kinase, interfere with neurexin binding and neurexin-induced oligomerization.

Mutations in the X-linked gene coding for the calcium-/calmodulin-dependent serine protein kinase (CASK) are associated with severe neurological disorders ranging from intellectual disability (in males) to mental retardation and microcephaly with pontine and cerebellar hypoplasia. CASK is involved in transcription control, in the regulation of trafficking of the post-synaptic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and acts as a presynaptic scaffolding protein. For CASK missense mutations, it is mostly unclear which of CASK's molecular interactions and cellular functions are altered and contribute to patient phenotypes. We identified five CASK missense mutations in male patients affected by neurodevelopmental disorders. These and five previously reported mutations were systematically analysed with respect to interaction with CASK interaction partners by co-expression and co-immunoprecipitation. We show that one mutation in the L27 domain interferes with binding to synapse-associated protein of 97 kDa. Two mutations in the guanylate kinase (GK) domain affect binding of CASK to the nuclear factors CASK-interacting nucleosome assembly protein (CINAP) and T-box, brain, 1 (Tbr1). A total of five mutations in GK as well as PSD-95/discs large/ZO-1 (PDZ) domains affect binding of CASK to the pre-synaptic cell adhesion molecule Neurexin. Upon expression in neurons, we observe that binding to Neurexin is not required for pre-synaptic localization of CASK. We show by bimolecular fluorescence complementation assay that Neurexin induces oligomerization of CASK, and that mutations in GK and PDZ domains interfere with the Neurexin-induced oligomerization of CASK. Our data are supported by molecular modelling, where we observe that the cooperative activity of PDZ, SH3 and GK domains is required for Neurexin binding and oligomerization of CASK.

The interaction between CASK and the tumour suppressor Dlg1 regulates mitotic spindle orientation in mammalian epithelia.

Oriented cell divisions are important for the formation of normal epithelial structures. Dlg1, a tumour suppressor, is required for mitotic spindle orientation in Drosophila epithelia and chick neuroepithelia, but how Dlg1 is localised to the membrane and its importance in mammalian epithelia are unknown. We show that Dlg1 is required in non-transformed mammalian epithelial cells for oriented cell divisions and normal lumen formation. We demonstrate that the MAGUK protein CASK, a membrane-associated scaffold, is the factor responsible for Dlg1 membrane localisation during spindle orientation, thereby identifying a new cellular function for CASK. Depletion of CASK leads to misoriented divisions in 3D, and to the formation of multilumen structures in cultured kidney and breast epithelial cells. Blocking the CASK-Dlg1 interaction with an interfering peptide, or by deletion of the CASK-interaction domain of Dlg1, disrupts spindle orientation and causes multilumen formation. We show that the CASK-Dlg1 interaction is important for localisation of the canonical LGN-NuMA complex known to be required for spindle orientation. These results establish the importance of the CASK-Dlg1 interaction in oriented cell division and epithelial integrity.This article has an associated First Person interview with the first author of the paper.

CASK regulates Notch pathway and functions as a tumor promoter in pancreatic cancer.

Calcium/calmodulin-dependent serine protein kinase (CASK), a member of membrane-associated guanylate kinase (MAGUK) super-family, is implicated in regulating cell proliferation, cytoskeletal remodeling, and cell metastasis. Our study aimed to investigate the effect of CASK on the malignant behaviors of pancreatic cancer cells and to determine the signaling pathway involved. CASK expression in pancreatic cancer tissues based on the TCGA database was analyzed using GEPIA online tool. The overall survival (OS) and disease-free survival (DFS) in patients with pancreatic cancer based on CASK expression was also analyzed using GEPIA. KEGG pathway enrichment analysis was used to show the association of 1522 CASK-related genes and signaling pathways. The expression of CASK, Notch1 and Hey1 was detected by Western blot. Cell proliferation, colony number, invasion, and apoptosis were detected by CCK-8, colony formation assay, Transwell invasion assay, and flow cytometry analysis, respectively. Results showed that CASK was upregulated in pancreatic cancer tissues and cells. Pancreatic cancer patients with high CASK expression showed shorter OS and DFS than patients with low CASK expression. KEGG pathway enrichment analysis proved that CASK and 1522 CASK-associated genes were primarily associated with the Notch pathway. CASK silencing inhibited cell proliferation, colony formation ability, and invasion and elicited apoptosis in pancreatic cancer cells. Additionally, we confirmed that CASK silencing inhibited the Notch pathway in pancreatic cancer cells. Overexpression of Notch1 resisted the anti-tumor functions of CASK knockdown in pancreatic cancer cells. In conclusion, CASK knockdown suppressed the malignant behaviors of pancreatic cancer cells by inactivating the Notch pathway.

Exploration of the Hsa-miR-1587-Protein Interaction and the Inhibition to CASK.

Hsa-miR-1587 has been found to be capable of forming G-quadruplex structures and is overexpressed in multiple cancer cell lines. Here, we explored the interactions between miR-1587 and proteins. HuProt™ human proteome microarray was utilized to screen the binding proteins, and it was discovered that CASK could bind to miR-1587 on the base of the G-quadruplex structure. Moreover, reelin and p21, which are downstream of CASK, were downregulated both transcriptionally and translationally by miR-1587, uncovered by q-RT-PCR and Western blot assays. Bioinformatic analysis was performed on STRING and Panther platforms, leading to the discovery that miR-1587 may be involved in intracellular metabolic and transcriptional physiological processes. This study explores the interaction of hsa-miR-1587 with proteins and provides a new strategy for the regulation of G-rich microRNA's function.

A German version of the Caregiver Skills scale for caregivers of patients with anorexia nervosa.

OBJECTIVE: To investigate acceptance, reliability, convergent validity, factor structure and sensitivity to change of a German translation of the Caregiver Skills (CASK) scale measuring skills related to caring for patients with eating disorders. METHODS: Two hundred and thirty-three parents (76% female) of adolescent patients (mean age 15.1) with anorexia nervosa (AN) completed the 27 items of the CASK. We calculated item/scale characteristics, internal consistencies and bivariate correlations with other measures of caregiving burden. We evaluated goodness-of-fit of the 6-factor model using confirmatory factors analysis and explored the sensitivity to change following two skills-based trainings. RESULTS: The fit of the 6-factor model was acceptable (Root Mean Square Error of Approximation: 0.077, Standard Root Mean Square Residual: 0.080). Cronbach's alpha was excellent for the total (.94) and acceptable for all subscales (0.73-0.85). The total CASK score was 68.04 (max. 100) showing relatively high self-rated caregiver skills. Non-completion rates of most items were low (<3%) indicating high acceptance. Convergent validity was found with measures of psychological distress, depression, anxiety and expressed emotion. The total score significantly increased following an 8-week workshop/online skills training (d = 0.70) and a 2-day multi-family intervention (d = 0.47). DISCUSSION: The German CASK version is a useful instrument to assess caregiver skills in parents of patients with AN and to evaluate outcomes of skills-based trainings.

Interactions between amyloid precursor protein-like (APPL) and MAGUK scaffolding proteins contribute to appetitive long-term memory in Drosophila melanogaster.

Amyloid precursor protein (APP), the precursor of amyloid beta peptide, plays a central role in Alzheimer's disease (AD), a pathology characterized by memory decline and synaptic loss upon aging. Understanding the physiological role of APP is fundamental in deciphering the progression of AD, and several studies suggest a synaptic function via protein-protein interactions. Nevertheless, it remains unclear whether and how these interactions contribute to memory. In Drosophila, we previously showed that APP-like (APPL), the fly APP homolog, is required for aversive associative memory in the olfactory memory center, the mushroom body (MB). In the present study, we show that APPL is required for appetitive long-term memory (LTM), another form of associative memory, in a specific neuronal subpopulation of the MB, the α'/ß' Kenyon cells. Using a biochemical approach, we identify the synaptic MAGUK (membrane-associated guanylate kinase) proteins X11, CASK, Dlgh2 and Dlgh4 as interactants of the APP intracellular domain (AICD). Next, we show that the Drosophila homologs CASK and Dlg are also required for appetitive LTM in the α'/ß' neurons. Finally, using a double RNAi approach, we demonstrate that genetic interactions between APPL and CASK, as well as between APPL and Dlg, are critical for appetitive LTM. In summary, our results suggest that APPL contributes to associative long-term memory through its interactions with the main synaptic scaffolding proteins CASK and Dlg. This function should be conserved across species.

Bioinformatics combined with quantitative proteomics analyses and identification of potential biomarkers in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is an invasive malignancy arising from biliary epithelial cells; it is the most common primary tumour of the bile tract and has a poor prognosis. The aim of this study was to screen prognostic biomarkers for CCA by integrated multiomics analysis. METHODS: The GSE32225 dataset was derived from the Gene Expression Omnibus (GEO) database and comprehensively analysed by using R software and The Cancer Genome Atlas (TCGA) database to obtain the differentially expressed RNAs (DERNAs) associated with CCA prognosis. Quantitative isobaric tags for relative and absolute quantification (iTRAQ) proteomics was used to screen differentially expressed proteins (DEPs) between CCA and nontumour tissues. Through integrated analysis of DERNA and DEP data, we obtained candidate proteins APOF, ITGAV and CASK, and immunohistochemistry was used to detect the expression of these proteins in CCA. The relationship between CASK expression and CCA prognosis was further analysed. RESULTS: Through bioinformatics analysis, 875 DERNAs were identified, of which 10 were associated with the prognosis of the CCA patients. A total of 487 DEPs were obtained by using the iTRAQ technique. Comprehensive analysis of multiomics data showed that CASK, ITGAV and APOF expression at both the mRNA and protein levels were different in CCA compared with nontumour tissues. CASK was found to be expressed in the cytoplasm and nucleus of CCA cells in 38 (45%) of 84 patients with CCA. Our results suggested that patients with positive CASK expression had significantly better overall survival (OS) and recurrence-free survival (RFS) than those with negative CASK expression. Univariate and multivariate analyses demonstrated that negative expression of CASK was a significantly independent risk factor for OS and RFS in CCA patients. CONCLUSIONS: CASK may be a tumour suppressor; its low expression is an independent risk factor for a poor prognosis in CCA patients, and so it could be used as a clinically valuable prognostic marker.

An N-terminal heterozygous missense CASK mutation is associated with microcephaly and bilateral retinal dystrophy plus optic nerve atrophy.

Heterozygous loss-of-function mutations in the X-linked gene CASK are associated with mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH) and ophthalmological disorders including optic nerve atrophy (ONA) and optic nerve hypoplasia (ONH). Recently, we have demonstrated that CASK(+/-) mice display ONH with 100% penetrance but exhibit no change in retinal lamination or structure. It is not clear if CASK loss-of-function predominantly affects retinal ganglion cells, or if other retinal cells like photoreceptors are also involved. Here, we report a heterozygous missense mutation in the N-terminal calcium/calmodulin-dependent kinase (CaMK) domain of the CASK protein in which a highly conserved leucine is mutated to the cyclic amino acid proline. In silico analysis suggests that the mutation may produce destabilizing structural changes. Experimentally, we observe pronounced misfolding and insolubility of the CASKL209P protein. Interestingly, the remaining soluble mutant protein fails to interact with Mint1, which specifically binds to CASK's CaMK domain, suggesting a mechanism for the phenotypes observed with the CASKL209P mutation. In addition to microcephaly, cerebellar hypoplasia and delayed development, the subject with the L209P mutation also presented with bilateral retinal dystrophy and ONA. Electroretinography indicated that rod photoreceptors are the most prominently affected cells. Our data suggest that the CASK interactions mediated by the CaMK domain may play a crucial role in retinal function, and thus, in addition to ONH, individuals with mutations in the CASK gene may exhibit other retinal disorders, depending on the nature of mutation.

PPAR-γ activation increases insulin secretion independent of CASK in INS-1 cells.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is expressed in pancreatic ß cells and is involved in insulin secretion. However, the precise mechanisms remain unclear. Calcium/calmodulin-dependent serine protein kinase (CASK), which plays a vital role in the anchoring of insulin granules on pancreatic ß cell membrane, is probably a downstream of the transcription factor PPAR-γ. The aim of the present study was to investigate the correlation among PPAR-γ, CASK and insulin secretion. We found that rosiglitazone (RSG) had a positive effect on the expression of CASK and PPAR-γ in INS-1 cells as shown by real-time polymerase chain reaction (PCR) and western blot analysis, but did not change the cellular location of CASK as shown by immunofluorescence assay. Knockdown of PPAR-γ significantly attenuated the mRNA and protein expression levels of CASK. ChIP-qPCR and luciferase assays showed that PPAR-γ bound with the Cask promoter, and promoter activity of Cask was elevated by RSG. RSG significantly enhanced the insulin secretion with potassium stimulation, but did not alter the insulin content as shown by potassium-stimulated insulin secretion assay. In addition, with RSG pretreatment, knockdown of Cask did not significantly affect the PPAR-γ activation-mediated insulin secretion. Moreover, electron microscopy demonstrated that with RSG pretreatment, silence of Cask did not change the number of vesicles anchored on the cell membranes compared with those in siCask-treated cells. Overall, the present study identifies that CASK is one of the PPAR-γ downstream targets and PPAR-γ exerts a positive effect on the expression of CASK in INS-1 cells. PPAR-γ activation increases insulin secretion independent of the upregulation of CASK.

Identification and functional characterization of a novel missense mutation in FRMD7 responsible for idiopathic congenital nystagmus.

Idiopathic congenital nystagmus (ICN) is a genetically heterogeneous eye movement disorder which seriously reduces childhood visual acuity. X-linked inheritance is the most common pattern, and mutations in FERM domain-containing protein 7 (FRMD7) are the major cause. Here, we recruited a four-generation Chinese family with X-linked ICN for the causative mutational screening of FRMD7. A novel missense variant, c.805 A > C, was identified in the proband. The mutation was confirmed in all the affected individuals but was not detected in unaffected family members or 100 unrelated Chinese male controls. The mutation causes a substitution of lysine to glutamine at position 269 (p.Lys269Gln, K269Q). The FRMD7 mutant inhibits the formation and extension of neurites. Moreover, the mutation disrupts FRMD7 interaction with calcium/calmodulin-dependent serine protein kinase and neurite formation. Together, our data expand the mutation spectrum of FRMD7 causing ICN and provide an insight into the pathogenesis of nystagmus.

Identification and Characterization of LIN-2(CASK) as a Regulator of Kinesin-3 UNC-104(KIF1A) Motility and Clustering in Neurons.

Kinesin-3 UNC-104(KIF1A) is the major axonal transporter of synaptic vesicles. Employing yeast two-hybrid and co-immunoprecipitation (Co-IP) assays, we characterized a LIN-2(CASK) binding site overlapping with that of reported UNC-104 activator protein SYD-2(Liprin-α) on the motor's stalk domain. We identified the L27 and GUK domains of LIN-2 to be the most critical interaction domains for UNC-104. Further, we demonstrated that the L27 domain interacts with the sterile alpha motifs (SAM) domains of SYD-2, while the GUK domain is able to interact with both the coiled coils and SAM domains of SYD-2. LIN-2 and SYD-2 colocalize in Caenorhabditis elegans neurons and display interactions in bimolecular fluorescence complementation (BiFC) assays. UNC-104 motor motility and Synaptobrevin-1 (SNB-1) cargo transport are largely diminished in neurons of LIN-2 knockout worms, which cannot be compensated by overexpressing SYD-2. The absence of the motor-activating function of LIN-2 results in increased motor clustering along axons, thus retaining SNB-1 cargo in cell bodies. LIN-2 and SYD-2 both positively affect the velocity of UNC-104, however, only LIN-2 is able to efficiently elevate the motor's run lengths. From our study, we conclude that LIN-2 and SYD-2 act in a functional complex to regulate the motor with LIN-2 being the more prominent activator.