Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells

Navone, Francesca; Niclas, Joshua; Hom-Booher, Nora; Sparks, Lowell L.; Bernstein, HD; McCaffrey, Gretchen; Vale, R. D.

doi:10.1083/jcb.117.6.1263

Cited by 188 publications

(161 citation statements)

References 67 publications

(88 reference statements)

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“…This band was absent from IPs containing GFP alone. Scale-up of the IPs and analysis by mass spectrometry revealed that the 120-kDa band contained Kif5B, the heavy-chain subunit of kinesin-1 [also known as conventional kinesin or KIF5 (32)]. Parallel yeast two-hybrid analysis (33), using a membrane proximal region of Nesp4 (residues 129-339) as bait, was performed by Myriad Genetics.…”

Section: Resultsmentioning

confidence: 99%

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization

Roux

Crisp

Liu

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

319

348

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Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin-Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1-3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning.centrosome ͉ LINC complex ͉ nuclear envelope T he nuclear envelope (NE) forms the interface between the nucleus and cytoplasm acting as a selective barrier that regulates nucleo-cytoplasmic traffic (1). In addition to this partition function, accumulating evidence reveals an essential role for the NE as a determinant of higher-order nuclear and chromatin organization (2). More surprising are findings that the NE directly impacts cytoskeletal architecture and in this way may help define the mechanical properties of the cell as a whole (3). The molecular bases for these effects are now emerging, uniting aspects of cellular physiology from mechanotransduction to nuclear positioning during differentiation and development (4).The NE is assembled from several elements, the most prominent being inner nuclear membranes (INMs) and outer nuclear membranes (ONMs) separated by a Ϸ40-nm gap or perinuclear space (PNS). The INM and ONM are joined where they are spanned by nuclear pore complexes (NPCs), the mediators of trafficking across the NE. The ONM also displays connections to the peripheral endoplasmic reticulum (ER). Accordingly, the INM, ONM, and ER represent a single membrane system with the PNS forming an extension of the ER lumen.The final feature of the NE is the nuclear lamina, a protein meshwork composed primarily of A-and B-type lamins that lines the nuclear face of the INM (2). The lamina is required for NE integrity and provides chromatin-anchoring sites at the nuclear periphery. Remarkably, aberrant A-type lamin expression, which is linked to several human diseases (5), is associated with altered cytoskeletal mechanics (3). The mechanisms underlying this phenomenon represent an intriguing biological problem.At least 60 NE membrane proteins are known (6). Although most likely reside in the INM, several ONM proteins have been identified (7). These include members of the mammalian nesprin (or syne) family (8, 9), Klarsicht (10) and 12) in Drosophila melanogaster, Anc-1 (13), and 16) in Caenorhabditis elegans, and Kms2 in the fission yeast Schizosaccharomyces pombe (17). A property that each of these has in common is that they interact with cytoskeletal components. They are also united in possessing a...

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Section: Resultsmentioning

confidence: 99%

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization

Roux

Crisp

Liu

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

319

348

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“…The C-terminal kinesin-1 tail can bind ribonucleoprotein cargoes (26) and has a nucleotide-independent MT-binding activity that may facilitate kinesin-1's MT transport/sliding function (27)(28)(29)(30). All these distinct apparatuses are located near the critical autoregulatory region of the KHC tail.…”

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confidence: 99%

“…The tail HM site can also bind MTs independent of the head (28). It has been suggested that MT binding by the tail allows kinesin-1 to slide and organize MT arrays (27,29,30).…”

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confidence: 99%

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Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

Wong

Rice

2010

Proc. Natl. Acad. Sci. U.S.A.

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Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules. Here, we present a role for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCs reduce the affinity of the head-tail interaction over tenfold and concomitantly repress the tail’s regulatory activity. We also show that KLCs inhibit tail-microtubule binding by a separate mechanism. Inhibition of head-tail binding requires steric and electrostatic factors. Inhibition of tail-microtubule binding is largely electrostatic, pH dependent, and mediated partly by a highly negatively charged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model wherein KLCs promote activation of kinesin-1 for cargo transport by simultaneously suppressing tail-head and tail-microtubule interactions. KLC-mediated inhibition of tail-microtubule binding may also influence diffusional movement of kinesin-1 on microtubules, and kinesin-1’s role in microtubule transport/sliding.

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“…Neuronal kinesin heavy chain (nKHC) is encoded by a gene distinct from the one specifying the ubiquitous kinesin heavy chain (uKHC), although the two proteins share a 65% amino acid sequence identity and a very similar domain organization (Niclas et al, 1994). Both their molecular structures show an N-terminal motor domain (Yang et al, 1989;Nakata and Hirokawa, 1995), an a-helical coiled coil rod (De Cuevas et al, 1992), and a small Cterminal globular domain that, despite its still unclear function, is thought to be involved in the interaction with intracellular cargo (Navone et al, 1992;Toyoshima et al, 1992;Andrews et al, 1993;Skoufias et al, 1994;Yu et al, 1995). The sequence of nKHC has the unique feature of a 70-amino-acid C-terminal stretch that is absent in the uKHC sequence (Niclas et al, 1994).…”

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confidence: 99%

Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat

Vignali

Lizier²,

Sprocati

et al. 1997

Journal of Neurochemistry

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Abstract:The kinesin family of motor proteins comprises at least two isoforms of conventional kinesin encoded by different genes: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, expressed exclusively in neuronal cells. In the present study, we have analyzed the expression of the two kinesin isoforms by immunochemistry at different stages of development of the rat CNS. We have found that the level of expression of neuronal kinesin is five to eight times higher in developing than in adult rat brains, whereas that of ubiquitous kinesin is only~2.5 times higher in maturing versus adult brains. Moreover, we have studied the distribution of neuronal kinesin by light microscopic immunocytochemistry in the rat brain at different postnatal ages and have found this protein not only to be more highly expressed in juvenile than in adult rat brains but also to show a different pattern of distribution. In particular, tracts of axonal fibers were clearly stained at early postnatal stages of development but were markedly unlabeled in adult rat brains. Our results indicate that the expression of at least one isoform of conventional neuron-specific kinesin is up-regulated in the developing rat CNS and suggest that this protein might play an important role in microtubule-based transport during the maturation of neuronal cells in vivo. Key Words: Microtubule-based motors-Conventional kinesin isoforms-Quantitative protein expression-Immunocytochemical localization-Brain development.

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Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells

Cited by 188 publications

References 67 publications

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization

Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat

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