Defective kinesin heavy chain behavior in mouse kinesin light chain mutants.

Conventional kinesin, kinesin-I, is a heterotetramer of two kinesin heavy chain (KHC) subunits (KIF5A, KIF5B, or KIF5C) and two kinesin light chain (KLC) subunits. While KHC contains the motor activity, the role of KLC remains unknown. It has been suggested that KLC is involved in either modulation of KHC activity or in cargo binding. Previously, we characterized KLC genes in mouse (Rahman, A., D.S. Friedman, and L.S. Goldstein. 1998. J. Biol. Chem. 273:15395-15403). Of the two characterized gene products, KLC1 was predominant in neuronal tissues, whereas KLC2 showed a more ubiquitous pattern of expression. To define the in vivo role of KLC, we generated KLC1 gene-targeted mice. Removal of functional KLC1 resulted in significantly smaller mutant mice that also exhibited pronounced motor disabilities. Biochemical analyses demonstrated that KLC1 mutant mice have a pool of KIF5A not associated with any known KLC subunit. Immunofluorescence studies of sensory and motor neuron cell bodies in KLC1 mutants revealed that KIF5A colocalized aberrantly with the peripheral cis-Golgi marker giantin in mutant cells. Striking changes and aberrant colocalization were also observed in the intracellular distribution of KIF5B and beta'-COP, a component of COP1 coatomer. Taken together, these data best support models that suggest that KLC1 is essential for proper KHC activation or targeting.     

Kinesin light-chain KLC3 expression in testis is restricted to spermatids

Kinesins are tetrameric motor molecules, consisting of two kinesin heavy chains (KHCs) and two kinesin light chains (KLCs) that are involved in transport of cargo along microtubules. The function of the light chain may be in cargo binding and regulation of kinesin activity. In the mouse, two KLC genes, KLC1 and KLC2, had been identified. KLC1 plays a role in neuronal transport, and KLC2 appears to be more widely expressed. We report the cloning from a testicular cDNA expression library of a mammalian light chain, KLC3. The KLC3 gene is located in close proximity to the ERCC2 gene. KLC3 can be classified as a genuine light chain: it interacts in vitro with the KHC, the interaction is mediated by a conserved heptad repeat sequence, and it associates in vitro with microtubules. In mouse and rat testis, KLC3 protein expression is restricted to round and elongating spermatids, and KLC3 is present in sperm tails. In contrast, KLC1 and KLC2 can only be detected before meiosis in testis. Interestingly, the expression profiles of the three known KHCs and KLC3 differ significantly: Kif5a and Kif5b are not expressed after meiosis, and Kif5c is expressed at an extremely low level in spermatids but is not detectable in sperm tails. Our characterization of the KLC3 gene suggests that it carries out a unique and specialized role in spermatids.     

Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms

Vaccinia virus (VACV) utilizes microtubule‐mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin‐1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin‐1, yet in its absence VACV egress still occurs on microtubules. During a co‐immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C‐terminal tail of KLC2, to a region that overlaps the binding site of cellular 14‐3‐3 proteins. F12/E2 displaces 14‐3‐3 from KLC and, unlike 14‐3‐3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N‐terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co‐operatively enhance A36 association with KLC, particularly when using a KLC1‐KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co‐operatively associate with kinesin‐1.      

A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome

Transport of cargoes by kinesin-1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit kinesin-1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define kinesin-1 binding. We now show that a bipartite tryptophan-based kinesin-1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for kinesin-1-dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan-based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases.     

Axonal transport of amyloid precursor protein is mediated by direct binding to the kinesin light chain subunit of kinesin-I

We analyzed the mechanism of axonal transport of the amyloid precursor protein (APP), which plays a major role in the development of Alzheimer's disease. Coimmunoprecipitation, sucrose gradient, and direct in vitro binding demonstrated that APP forms a complex with the microtubule motor, conventional kinesin (kinesin-I), by binding directly to the TPR domain of the kinesin light chain (KLC) subunit. The estimated apparent Kd for binding is 15-20 nM, with a binding stoichiometry of two APP per KLC. In addition, association of APP with microtubules and axonal transport of APP is greatly decreased in a gene-targeted mouse mutant of the neuronally enriched KLC1 gene. We propose that one of the normal functions of APP may be as a membrane cargo receptor for kinesin-I and that KLC is important for kinesin-I-driven transport of APP into axons.     

Cargo selection by specific kinesin light chain 1 isoforms

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures.     

A molecular dynamics study of the binary complexes of APP,JIP1, and the cargo binding domain of KLC

Mutations in the amyloid precursor protein (APP) are responsible for the formation of amyloid‐β peptides. These peptides play a role in Alzheimer's and other dementia‐related diseases. The cargo binding domain of the kinesin‐1 light chain motor protein (KLC1) may be responsible for transporting APP either directly or via interaction with C‐jun N‐terminal kinase‐interacting protein 1 (JIP1). However, to date there has been no direct experimental or computational assessment of such binding at the atomistic level. We used molecular dynamics and free energy estimations to gauge the affinity for the binary complexes of KLC1, APP, and JIP1. We find that all binary complexes (KLC1:APP, KLC1:JIP1, and APP:JIP1) contain conformations with favorable binding free energies. For KLC1:APP the inclusion of approximate entropies reduces the favorability. This is likely due to the flexibility of the 42‐residue APP protein. In all cases we analyze atomistic/residue driving forces for favorable interactions. Proteins 2017; 85:221–234. © 2016 Wiley Periodicals, Inc.     

JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation

Kinesin-1 is a molecular motor responsible for cargo transport along microtubules and plays critical roles in polarized cells, such as neurons. Kinesin-1 can function as a dimer of two kinesin heavy chains (KHC), which harbor the motor domain, or as a tetramer in combination with two accessory light chains (KLC). To ensure proper cargo distribution, kinesin-1 activity is precisely regulated. Both KLC and KHC subunits bind cargoes or regulatory proteins to engage the motor for movement along microtubules. We previously showed that the scaffolding protein JIP3 interacts directly with KHC in addition to its interaction with KLC and positively regulates dimeric KHC motility. Here we determined the stoichiometry of JIP3-KHC complexes and observed approximately four JIP3 molecules binding per KHC dimer. We then determined whether JIP3 activates tetrameric kinesin-1 motility. Using an in vitro motility assay, we show that JIP3 binding to KLC engages kinesin-1 with microtubules and that JIP3 binding to KHC promotes kinesin-1 motility along microtubules. We tested the in vivo relevance of these findings using axon elongation as a model for kinesin-1-dependent cellular function. We demonstrate that JIP3 binding to KHC, but not KLC, is essential for axon elongation in hippocampal neurons as well as axon regeneration in sensory neurons. These findings reveal that JIP3 regulation of kinesin-1 motility is critical for axon elongation and regeneration.     

Co-operative versus independent transport of different cargoes by Kinesin-1

Kinesin motors drive the intracellular transport of multiple cargoes along microtubule tracks; yet, how kinesins discriminate among their many potential cargoes is unknown. We tested whether Kinesin-1 cargoes compete, co-operate or are transported independently of each other. We focused on Kinesin-1 cargoes that bind directly to the kinesin light chain (KLC) subunit, namely the c-Jun NH₂-terminal kinase-interacting proteins (JIPs) 1 and 3, Kidins220/ARMS and PAT1. Overexpression of individual cargo proteins in differentiated CAD cells resulted in mislocalization of the endogenous protein but had no effect on localization of other cargo proteins to neurite tips. Thus, while transport of distinct cargoes is saturable, they do not compete with each other. Interestingly, we found that low expression of JIP1 or JIP3 enhanced the transport of the other JIP to neurite tips. Moreover, JIP1 and JIP3 require each other for transport. Co-operative transport is due to an interaction between JIP1 and JIP3 as well as distinct binding sites on the KLC tetratricopeptide repeat (TPR) bundle: the TPR groove binds to C-terminal residues of JIP1, whereas the TPR surface binds to internal residues in JIP3. Formation of a JIP1/JIP3/KLC complex is necessary for efficient JIP1 or JIP3 transport in neuronal cells. Thus, JIP scaffolding proteins are transported in a co-operative manner, despite the independent transport of other Kinesin-1 cargoes.     

Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility

Neuronal development, function and repair critically depend on axonal transport of vesicles and protein complexes, which is mediated in part by the molecular motor kinesin-1. Adaptor proteins recruit kinesin-1 to vesicles via direct association with kinesin heavy chain (KHC), the force-generating component, or via the accessory light chain (KLC). Binding of adaptors to the motor is believed to engage the motor for microtubule-based transport. We report that the adaptor protein Sunday Driver (syd, also known as JIP3 or JSAP1) interacts directly with KHC, in addition to and independently of its known interaction with KLC. Using an in vitro motility assay, we show that syd activates KHC for transport and enhances its motility, increasing both KHC velocity and run length. syd binding to KHC is functional in neurons, as syd mutants that bind KHC but not KLC are transported to axons and dendrites similarly to wild-type syd. This transport does not rely on syd oligomerization with itself or other JIP family members. These results establish syd as a positive regulator of kinesin activity and motility.     

A Specific Light Chain of Kinesin Associates with Mitochondria in Cultured Cells

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1–3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.     

Rat kinesin light chain 3 associates with spermatid mitochondria

We recently discovered that in rat spermatids, kinesin light chain KLC3 can associate with outer dense fibers, major sperm tail components, and accumulates in the sperm midpiece. Here, we show that mitochondria isolated from rat-elongating spermatids have bound KLC3. Immunoelectron microscopy indicates that the association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the plasma membrane to the developing midpiece. KLC3 is able to bind in vitro to mitochondria from spermatids as well as somatic cells employing a conserved kinesin light chain motif, the tetratrico-peptide repeats. Expression of KLC3 in fibroblasts results in formation of large KLC3 clusters close to the nucleus, which also contain mitochondria: no other organelles were present in these clusters. Mitochondria are not present in KLC3 clusters after deletion of KLC3's tetratrico-peptide repeats. Our results indicate that the rat spermatid kinesin light chain KLC3 can associate with mitochondria.     

Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules

The cargo that the molecular motor kinesin moves along microtubules has been elusive. We searched for binding partners of the COOH terminus of kinesin light chain, which contains tetratricopeptide repeat (TPR) motifs. Three proteins were found, the c-jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) JIP-1, JIP-2, and JIP-3, which are scaffolding proteins for the JNK signaling pathway. Concentration of JIPs in nerve terminals requires kinesin, as evident from the analysis of JIP COOH-terminal mutants and dominant negative kinesin constructs. Coprecipitation experiments suggest that kinesin carries the JIP scaffolds preloaded with cytoplasmic (dual leucine zipper-bearing kinase) and transmembrane signaling molecules (the Reelin receptor, ApoER2). These results demonstrate a direct interaction between conventional kinesin and a cargo, indicate that motor proteins are linked to their membranous cargo via scaffolding proteins, and support a role for motor proteins in spatial regulation of signal transduction pathways.     

Dynamin-1-like protein (Dnm1L) interaction with kinesin light chain 1 (KLC1) through the tetratricopeptide repeat (TPR) domains

Kinesin light chain 1 (KLC1) mediates binding of KIF5 motor to specific cargo. Using the yeast two-hybrid screening, we found that mitochondrial fission protein dynamin-1-like protein (Dnm1L) interacted with KLC1, but not KIF5. Dnm1L and KLC1 were co-localized in cultured cells. These results suggest that KLC1 may play a potential role in post-fission mitochondrial transport.     

Association of kinesin light chain with outer dense fibers in a microtubule-independent fashion

Conventional kinesin I motor molecules are heterotetramers consisting of two kinesin light chains (KLCs) and two kinesin heavy chains. The interaction between the heavy and light chains is mediated by the KLC heptad repeat (HR), a leucine zipper-like motif. Kinesins bind to microtubules and are involved in various cellular functions, including transport and cell division. We recently isolated a novel KLC gene, klc3. klc3 is the only known KLC expressed in post-meiotic male germ cells. A monoclonal anti-KLC3 antibody was developed that, in immunoelectron microscopy, detects KLC3 protein associated with outer dense fibers (ODFs), unique structural components of sperm tails. No significant binding of KLC3 with microtubules was observed with this monoclonal antibody. In vitro experiments showed that KLC3-ODF binding occurred in the absence of kinesin heavy chains or microtubules and required the KLC3 HR. ODF1, a major ODF protein, was identified as the KLC3 binding partner. The ODF1 leucine zipper and the KLC3 HR mediated the interaction. These results identify and characterize a novel interaction between a KLC and a non-microtubule macromolecular structure and suggest that KLC3 could play a microtubule-independent role during formation of sperm tails.     

KLC3 is involved in sperm tail midpiece formation and sperm function

Kinesin light chain 3 (KLC3) is the only known kinesin light chain expressed in post-meiotic male germ cells. We have reported that in rat spermatids KLC3 associates with outer dense fibers and mitochondrial sheath. KLC3 is able to bind to mitochondria in vitro and in vivo employing the conserved tetratrico-peptide repeat kinesin light chain motif. The temporal expression and association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the spermatid cell periphery to the developing midpiece suggesting a role in midpiece formation. In fibroblasts, expression of KLC3 results in formation of large KLC3 aggregates close to the nucleus that contain mitochondria. However, the molecular basis of the aggregation of mitochondria by KLC3 and its role in sperm tail midpiece formation are not clear. Here we show that KLC3 expression from an inducible system causes mitochondrial aggregation within 6h in a microtubule dependent manner. We identified the mitochondrial outer membrane porin protein VDAC2 as a KLC3 binding partner. To analyze a role for KLC3 in spermatids we developed a transgenic mouse model in which a KLC3ΔHR mutant protein is specifically expressed in spermatids: this KLC3 mutant protein binds mitochondria and causes aggregate formation, but cannot bind outer dense fibers. Male transgenic mice display significantly reduced reproductive efficiency siring small sized litters. We observed defects in the mitochondrial sheath structure in a number of transgenic spermatids. Transgenic males have a significantly reduced sperm count and produce spermatozoa that exhibit abnormal motility parameters. Our results indicate that KLC3 plays a role during spermiogenesis in the development of the midpiece and in the normal function of spermatozoa.     

Multisite and bidirectional exonic splicing enhancer in CD44 alternative exon v3

The human CD44 gene encodes multiple isoforms of a transmembrane protein that differ in their extracellular domains as a result of alternative splicing of its variable exons. Expression of CD44 is tightly regulated according to the type and physiological status of a cell, with expression of high molecular weight isoforms by inclusion of variable exons and low molecular weight isoforms containing few or no variable exons. Human CD44 variable exon 3 (v3) can follow a specific alternative splicing route different from that affecting other variable exons. Here we map and functionally describe the splicing enhancer element within CD44 exon v3 which regulates its inclusion in the final mRNA. The v3 splicing enhancer is a multisite bipartite element consisting of a tandem nonamer, the XX motif, and an heptamer, the Y motif, located centrally in the exon. Each of the three sites of this multisite enhancer partially retains its splicing enhancing capacity independently from each other in CD44 and shows full enhancing function in gene contexts different from CD44. We further demonstrate that these motifs act cooperatively as at least two motifs are needed to maintain exon inclusion. Their action is differential with respect to the splice-site target abutting v3. The first X motif acts on the 3' splice site, the second X motif acts on both splice sites (as a bidirectional exonic splicing enhancer), and the Y motif acts on the 5' splice site. We also show that the multisite v3 splicing enhancer is functional irrespective of flanking intron length and spatial organization within v3.     

鉴定9个新的RHD基因mRNA可变剪接体

为了研究各种RHD基因mRNA可变剪接体的基因结构, 应用逆转录聚合酶链反应(RT-PCR)检测正常人脐血样本RHD mRNA, 对RHD cDNA进行TA克隆和序列分析, 对各可变剪接体的剪接位点进行DNA序列分析, 并将RHD mRNA进行表达序列标签(ESTs)分析。结果在28个阳性克隆中, 除全长RHD cDNA外, 共检测到12种(包括9种新的)RHD可变剪接体, 发现外显子遗漏、5′和3′剪接位点变异3种剪接形式, 涉及外显子2~9, 其中6种新的剪接体同时存在RHD和RHCE基因同源杂交现象。ESTs分析还检索到内含子保留形式的剪接体。研究表明, RHD基因mRNA存在复杂的可变剪接机制, 除已报道的剪接体外, 检测到9种新的RHD可变剪接体, 并发现了可变剪接和同源杂交并存现象。