Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.
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PMID:Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle. 775 99

In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves. We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation.
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PMID:Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28. 888 67

We have identified a new member of the kinesin superfamily in Drosophila, KLP38B (kinesin-like protein at 38B). KLP38B was isolated through its two-hybrid interaction with the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1). We demonstrate that recombinant KLP38B and PP1 associate in vitro. This is the first demonstration of direct binding of a kinesin-related protein to a regulatory enzyme. Though most closely related to the Unc-104 subfamily of kinesin-related proteins, KLP38B is expressed only in proliferating cells. KLP38B mutants show cell proliferation defects in many tissues. KLP38B is required for normal chromatin condensation as embryos from KLP38B mutant mothers have undercondensed chromatin at metaphase and anaphase. This is the first time that a kinesin-related protein has been shown to have such a role. Incomplete lethality of a strong KLP38B allele suggests partial redundancy with one or more additional kinesin-related proteins.
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PMID:KLP38B: a mitotic kinesin-related protein that binds PP1. 923 81

The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase. To identify proteins able to interact with the tail of HsEg5, we performed a yeast two-hybrid screen with a HsEg5 stalk-tail construct as bait. We isolated a cDNA coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex. The interaction between HsEg5 and p150(Glued) was enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 had a phosphorylatable residue at position 927. Phosphorylation also enhanced the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy revealed co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors.
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PMID:Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150. 923 42

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive alpha-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 micron/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.
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PMID:A developmentally regulated kinesin-related motor protein from Dictyostelium discoideum. 969 69

Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr(291) results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin.
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PMID:A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor. 1076 90

Mitosis utilizes a number of kinesin-related proteins (KRPs). Here we report the identification of a novel KRP termed KRMP1, which has a deduced 1780-amino acid sequence composed of ternary domains. The amino-terminal head domain is most similar to the kinesin motor domain of the MKLP-1 subfamily and has an intrinsic ATPase activity that is diminished by substituting the consensus Lys-168 with Arg. The central stalk domain is predicted to form a long alpha-helical coiled-coil, and can interact with each other in vivo. An in vivo labeling experiment revealed that KRMP1 is phosphorylated, and we also found that the region within the tail domain containing Thr-1604 as the cdc2 kinase phosphorylation site differs from the bimC box conserved in the bimC subfamily of KRPs. Immunofluorescence analysis showed that endogenous KRMP1 was localized predominantly to the cytoplasm during interphase and dispersed throughout the cell during mitosis. Consistent with this finding, overexpressed KRMP1 was detected in a complicated nuclear or cytoplasmic pattern reflecting multiple nuclear localization/export signals. Furthermore, KRMP1 interacted with the mitotic peptidyl-prolyl isomerase Pin1 in vivo, and an in vitro interaction was detected between the tail domain of KRMP1 and the WW domain of Pin1. Overexpression of KRMP1 caused COS-7 cells to arrest at G(2)-M, and co-expression of Pin1 reversed this effect, indicating their physiological interaction. Together, our results suggest that KRMP1 is a mitotic target regulated by Pin1 and vice versa.
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PMID:Identification of a novel kinesin-related protein, KRMP1, as a target for mitotic peptidyl-prolyl isomerase Pin1. 1147 Aug 1

We have isolated a novel protein based on its association with Drosophila APP-like protein (APPL), a homolog of the beta-amyloid precursor protein (APP) that is implicated in Alzheimer's disease. This novel APPL-interacting protein 1 (APLIP1) contains a Src homology 3 domain and a phosphotyrosine interaction domain and is expressed abundantly in neural tissues. The phosphotyrosine interaction domain of APLIP1 interacts with a sequence containing GYENPTY in the cytoplasmic domain of APPL. APLIP1 is highly homologous to the carboxyl-terminal halves of mammalian c-Jun NH(2)-terminal kinase (JNK)-interacting protein 1b (JIP1b) and 2 (JIP2), which also contain Src homology 3 and phosphotyrosine interaction domains. The similarity of APLIP1 to JIP1b and JIP2 includes interaction with component(s) of the JNK signaling pathway and with the motor protein kinesin and the formation of homo-oligomers. JIP1b interacts strongly with the cytoplasmic domain of APP (APPcyt), as APLIP1 does with APPL, but the interaction of JIP2 with APPcyt is weak. Overexpression of JIP1b slightly enhances the JNK-dependent threonine phosphorylation of APP in cultured cells, but that of JIP2 suppresses it. These observations suggest that the interactions of APP family proteins with APLIP1, JIP1b, and JIP2 are conserved and play important roles in the metabolism and/or the function of APPs including the regulation of APP phosphorylation by JNK. Analysis of APP family proteins and their associated proteins is expected to contribute to understanding the molecular process of neural degeneration in Alzheimer's disease.
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PMID:Interaction of Alzheimer's beta -amyloid precursor family proteins with scaffold proteins of the JNK signaling cascade. 1191 89

Glucose stimulates the release of insulin in part by activating the recruitment of secretory vesicles to the cell surface. While this movement is known to be microtubule-dependent, the molecular motors involved are undefined. Active kinesin was found to be essential for vesicle translocation in live beta-cells, since microinjection of cDNA encoding dominant-negative KHC(mut) (motor domain of kinesin heavy chain containing a Thr(93)-->Asn point mutation) blocked vesicular movements. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Thus, vesicles released during the first phase of insulin secretion exist largely within a translocation-independent pool. Kinesin-driven anterograde movement of vesicles is then necessary for the sustained (second phase) of insulin release. Kinesin may, therefore, represent a novel target for increases in intracellular ATP concentrations in response to elevated extracellular glucose and may be involved in the ATP-sensitive K+channel-independent stimulation of secretion by the sugar.
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PMID:Molecular mechanisms involved in secretory vesicle recruitment to the plasma membrane in beta-cells. 1202 74

In an effort to understand how specific structural features within the kinesin neck, a region of the heavy chain located between the catalytic core and stalk domains, may contribute to motor processivity (an ability to remain attached to the microtubule filament), we have prepared several synthetic peptides corresponding to the neck region of human conventional kinesin and determined their secondary structure content and stability by CD spectroscopy. Our results show that the coiled-coil dimerization domain within the human kinesin neck region corresponds to residues 337 to 369 in solution, and thus is in excellent agreement with the recent X-ray crystallographic structures of rat brain kinesin. Further, we show that the first and last heptads of this region are absolutely critical for creating the high stability and association of the dimeric structure. Interestingly, addition of the 7 N-terminal neck-linker residues (330-336) to the coiled-coil domain significantly increased its stability (Delta GdnHCl midpoint of 1 M or an increase of approximately 1.5 kcal/mol), indicating that a strong structural link exists between the neck-linker and coiled-coil region. Subsequent high-resolution structural analysis of the residues located at the junction of the neck-linker and coiled-coil revealed the presence of the two helix capping motifs, the capping box (a reciprocal interaction of Thr 336 with Gln 339) and the hydrophobic staple (a hydrophobic packing interaction of Leu 335 with Trp 340). Substitution of Leu 335 and Thr 336 (the capping residues) with Gly completely eliminated the increased stability of the coiled-coil region observed in the presence of the neck-linker residues. Correspondingly, substitution of Trp 340, the first hydrophobic core d position residue of the coiled-coil, with an Ala residue resulted in a greater than expected decrease in stability and helicity of the coiled-coil structure. Subsequent analysis of the X-ray structure and substitution analysis of Lys 341 revealed that Trp 340 makes an important interchain hydrophobic interaction with Lys 341 of the opposite chain. Taken together these results reveal that a set of strong intra- and inter-chain interactions made up of the helix "capping box," "hydrophobic staple," and the newly identified "Leu-Trp-Lys sandwich" motifs stabilize the kinesin neck coiled-coil structure, thus preventing it from fraying and unfolding.
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PMID:Helix capping interactions stabilize the N-terminus of the kinesin neck coiled-coil. 1206 48


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