Novel 44-kilodalton subunit of axonemal Dynein conserved from chlamydomonas to mammals

Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.     

The “8-kD” Cytoplasmic Dynein Light Chain Is Required for Nuclear Migration and for Dynein Heavy Chain Localization in Aspergillus nidulans

The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. Here we report on a new gene required for nuclear migration, nudG, which encodes a homologue of the “8-kD” cytoplasmic dynein light chain (CDLC). We demonstrate that the temperature sensitive nudG8 mutation inhibits nuclear migration and growth at restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain (CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact.     

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.     

Identification of a novel dynein binding domain in nudel essential for spindle pole organization in Xenopus egg extract

The nuclear distribution protein E (NudE) and nuclear distribution protein E-like (Nudel or Ndel1) interact with both lissencephaly 1 (Lis1) and dynein. These interactions are thought to be essential for dynein function. Previous studies have shown that the highly conserved N terminus of NudE/Nudel directly binds to Lis1, and such binding is critical for dynein activity. By contrast, although the C terminus of NudE/Nudel was reported to bind to dynein, the functional significance of this binding has remained unclear. Using the sperm-mediated spindle assembly assay in Xenopus egg extracts and extensive mutagenesis studies, we have identified a highly conserved dynein binding domain within the first 80 amino acids of Nudel. We further demonstrate that the dynein intermediate chain in the dynein complex is directly involved in this interaction. Importantly, we show that both the dynein and Lis1 binding domains of Nudel are required for spindle pole organization. Finally, we report that spindle defects caused by immuno-depletion of Nudel could be rescued by a 1-fold increase of Lis1 concentration in Xenopus egg extracts. This suggests that an important function of the N terminus of Nudel is to facilitate the interaction between Lis1 and dynein during spindle assembly. Together, our findings open up new avenues to further decipher the mechanism of dynein regulation by Nudel and Lis1.     

Cyclic AMP inhibits p38 activation via CREB-induced dynein light chain

The mitogen-activated protein kinase p38 plays a critical role in inflammation, cell cycle progression, differentiation, and apoptosis. The activity of p38 is stimulated by a variety of extracellular stimuli, such as the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), and subjected to regulation by other intracellular signaling pathways, including the cyclic AMP (cAMP) pathway. Yet the underlying mechanism by which cAMP inhibits p38 activation is unknown. Here we show that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of p38 activation. cAMP inhibits p38 activation via the protein kinase A-CREB pathway. The inhibition is mediated by the CREB target gene Dlc, whose protein product, DLC, interferes with the formation of the MKK3/6-p38 complex, thereby suppressing p38 phosphorylation activation by MKK3/6. The inhibition of p38 activation by cAMP leads to suppression of NF-kappaB activity and promotion of apoptosis in response to TNF-alpha. Thus, our results identify DLC as a novel inhibitor of the p38 pathway and provide a molecular mechanism by which cAMP suppresses p38 activation and promotes apoptosis.     

Structural dynamics and multiregion interactions in dynein-dynactin recognition

Cytoplasmic dynein is a 1.2-MDa multisubunit motor protein complex that, together with its activator dynactin, is responsible for the majority of minus end microtubule-based motility. Dynactin targets dynein to specific cellular locations, links dynein to cargo, and increases dynein processivity. These two macromolecular complexes are connected by a direct interaction between dynactin's largest subunit, p150(Glued), and dynein intermediate chain (IC) subunit. Here, we demonstrate using NMR spectroscopy and isothermal titration calorimetry that the binding footprint of p150(Glued) on IC involves two noncontiguous recognition regions, and both are required for full binding affinity. In apo-IC, the helical structure of region 1, the nascent helix of region 2, and the disorder in the rest of the chain are determined from coupling constants, amide-amide sequential NOEs, secondary chemical shifts, and various dynamics measurements. When bound to p150(Glued), different patterns of spectral exchange broadening suggest that region 1 forms a coiled-coil and region 2 a packed stable helix, with the intervening residues remaining disordered. In the 150-kDa complex of p150(Glued), IC, and two light chains, the noninterface segments remain disordered. The multiregion IC binding interface, the partial disorder of region 2 and its potential for post-translational modification, and the modulation of the length of the longer linker by alternative splicing may provide a basis for elegant and multifaceted regulation of binding between IC and p150(Glued). The long disordered linker between the p150(Glued) binding segments and the dynein light chain consensus sequences could also provide an attractive recognition platform for diverse cargoes.     

Mechanisms regulating the nuclear translocation of p38 MAP kinase

p38 mitogen‐activated protein kinase (MAPK) is of fundamental importance in a cell's response to environmental stresses, cytokines and DNA damage. p38 resides in the cytoplasm of resting cells, and translocates into the nucleus upon activation, yet the exact mechanisms remain largely unclear. We show here that the phosphorylation‐dependent nuclear translocation of p38 is a common phenomenon when cells are stimulated with various stresses. On the other hand, the nuclear export of p38 requires its dephosphorylation, and it is exported both in a MK2‐dependent and a nuclear export signal (NES)‐independent manner. Although different p38‐regulated/activated protein kinase (PRAK) mutants all dictate the intracellular localization of p38, results from a PRAK‐deficient cell line indicate that it plays no role in this process. Microtubule depolymerizing reagent nocodazole and dynein inhibitor EHNA both block the nuclear translocation of p38, demonstrating roles for microtubules and dynein in p38 transport. Taken together, stress‐induced nuclear accumulation of p38 is a phosphorylation‐dependent, microtubule‐ and dynein‐associated process. J. Cell. Biochem. 110: 1420–1429, 2010. © 2010 Wiley‐Liss, Inc.     

p150(Glued), Dynein, and microtubules are specifically required for activation of MKK3/6 and p38 MAPKs

To look for regulators of the mitogen-activated protein kinase (MAPK) kinase 6 (MKK6), a yeast two-hybrid screen was initiated using MKK6 as bait. p150(Glued) dynactin, a key component of the cytoplasmic dynein-dynactin motor complex, was found to specifically interact with MKK6 and its close homologue MKK3. Silencing of p150(Glued) expression by small interference RNA reduced the stimulus-induced phosphorylation of MKK3/6 and p38 MAPKs. The similar adverse effect was also seen when the cytoplasmic dynein motor was disrupted by other means. Like p150(Glued), MKK3/6 directly associate with microtubules. Disruption of microtubules prior to cell stimulation specifically inhibits the stimulus-induced phosphorylation of both MKK3/6 and p38 MAPKs. Our unexpected findings reveal a specific requirement for p150(Glued)/dynein/functional microtubules in activation of MKK3/6 and p38 MAPKs in vivo.     

Regulation of 22S dynein by a 29-kD light chain

Previously, a 29-kD axonemal polypeptide (p29) that copurifies with 22S dynein has been shown to be phosphorylated in a cAMP- and Ca(2+)- sensitive manner, consistent with a role for this molecule in the signal transduction cascade leading to fast forward swimming in Paramecium tetraurelia (Hamasaki, T., K. Barkalow, J. Richmond, and P. Satir. 1991. Proc. Natl. Acad. Sci. USA. 88:7912-7922). This study demonstrates the nature of the relationship between p29 and 22S dynein. Chaotropic agents can be used to separate p29 fractions from 22S dynein. When extracted p29 is exchanged into physiological buffers, it regains the ability to recombine with 22S dynein with an apparent dissociation constant of 25 nM; no recombination is seen with 14S dynein or with unrelated control proteins. p29 from Paramecium will also recombine with Tetrahymena 22 but not 14S dynein. After chymotryptic digestion of 22S dynein, p29 preferentially binds to a single-headed fragment, homologous to the alpha H chain of Tetrahymena 22S dynein. 22S dynein treated in vitro by Paramecium protein kinase A in the presence of cAMP and ATP to phosphorylate p29 translocates bovine brain microtubules significantly (1.53x; p < 0.001) faster than before phosphorylation. Similarly, 22S dynein reconstituted in vitro with thiophosphorylated p29 translocates microtubules significantly (1.31x; p < 0.001) faster than controls reconstituted with nonthiophosphorylated p29. p29 is the only moiety thiophosphorylated in the reconstituted dynein. We conclude that p29 functions as a 22S dynein regulatory light chain in that it alone is sufficient to control the rate of microtubule translocation by changes in its phosphorylation state.     

The 78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein

A previous study (King et al., 1991. J. Biol. Chem. 266:8401-8407) showed that the 78,000-M(r) intermediate chain (IC78) from the Chlamydomonas outer arm dynein is in direct contact with alpha-tubulin in situ, suggesting that this protein may be involved in binding the dynein to the doublet microtubules. Molecular genetic analysis of this chain recently demonstrated that it is a WD repeat protein essential for outer arm assembly (Wilkerson et al., 1995.J. Cell Biol. 129:169- 178). We have now transcribed and translated IC78 in vitro, and demonstrate that this molecule binds axonemes and microtubules, whereas a homologous protein (the 69,000-M(r) intermediate chain [IC69] of Chlamydomonas outer arm dynein) does not. Thus, IC78 is a bona fide microtubule-binding protein. Taken together with the previous results, these findings indicate that IC78 is likely to provide at least some of the adhesive force that holds the dynein to the doublet microtubule, and support the general hypothesis that the dynein intermediate chains are involved in targeting different dyneins to the specific cell organelles with which they associate. Analysis of the binding activities of various IC78 deletion constructs translated in vitro identified discrete regions of IC78 that affected the binding to microtubules; two of these regions are specifically missing in IC69. Previous studies also showed that IC78 is in direct contact with IC69; the current work indicates that the region of IC78 that mediates this interaction is coincident with two of IC78's WD repeats. This supports the hypothesis that these repeats are involved in protein-protein interactions within the dynein complex.     

Intrinsic disorder in dynein intermediate chain modulates its interactions with NudE and dynactin

The functional diversity of cytoplasmic dynein is in part attributed to multiple interactions between noncatalytic dynein subunits and an array of regulatory proteins. This study focuses on the interaction between the dynein intermediate chain subunit (IC) and a dynein regulator protein (NudE). We use isothermal titration calorimetry and NMR spectroscopy to map their interacting sections to their respective N-terminal domains, which are predicted to form dimeric coiled-coils. Interestingly, the specific residues within IC that interact with NudE are a subset of the bi-segmental binding region reported for p150(Glued), a subunit of the dynein activator protein dynactin. Although the IC binding domains of both NudE and p150(Glued) form dimeric coiled-coils and bind IC at a common site, we observe distinct binding modes for each regulatory protein: 1) NudE binds region 1 of the bi-segmental binding footprint of p150(Glued), whereas p150(Glued) requires regions 1 and 2 to match the binding affinity of NudE with region 1 alone. 2) Compared with unbound IC, NudE-bound IC shows a slight increase in flexibility in region 2, in contrast to the increase in ordered structure observed for p150(Glued)-bound IC (Morgan, J. L., Song, Y., and Barbar, E. (2011) J. Biol. Chem. 286, 39349-39359). 3) Although NudE has a higher affinity for the common binding segment on IC, when all three proteins are in solution, IC preferentially binds p150(Glued). These results underscore the importance of a bi-segmental binding region of IC and disorder in region 2 and flanking linkers in selecting which regulatory protein binds IC.     

Novel role of cytoplasmic dynein motor in maintenance of the nuclear number in conidia through organized conidiation in Aspergillus oryzae

Cytoplasmic dynein is a minus-end-directed, microtubule-dependent motor protein complex. DhcA, cytoplasmic dynein heavy chain in Aspergillus oryzae, contained four P-loops involved in ATP binding which were conserved as in cytoplasmic dynein heavy chains of other organisms. The amino acid sequence of A. oryzae DhcA was similar to cytoplasmic dynein heavy chains from other organisms except for the N-terminus of Saccharomyces cerevisiae Dyn1. Disruption of dhcA gene in the region encoding four P-loop motifs resulted in a defective growth and perturbed distribution of nuclei and vacuoles. The dhcA disruptant exhibited an abnormal morphology of conidial heads and conidia with an increased nuclear number. The present study implicates a novel role of cytoplasmic dynein in maintenance of the nuclear number in conidia through an organized conidiation.     

A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.     

The DYNLT3 light chain directly links cytoplasmic dynein to a spindle checkpoint protein, Bub3

Cytoplasmic dynein is the motor protein responsible for the intracellular transport of various organelles and other cargoes toward microtubule minus ends. However, it remains to be determined how dynein is regulated to accomplish its varied roles. The dynein complex contains six subunits, including three classes of light chains. The two isoforms of the DYNLT (Tctex1) family of light chains, DYNLT1 and DYNLT3, have been proposed to link dynein to specific cargoes. However, no specific binding partner had been found for the DYNLT3 light chain. We find that DYNLT3 binds to Bub3, a spindle checkpoint protein. Bub3 binds exclusively to DYNLT3 and not to the other dynein light chains. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that Bub3 interacts with the cytoplasmic dynein complex. DYNLT3 is present on kinetochores at prometaphase, but not later mitotic stages, demonstrating that this dynein light chain, like Bub3 and other checkpoint proteins, is depleted from the kinetochore during chromosome alignment. Knockdown of DYNLT3 with small interference RNA increases the mitotic index, in particular, the number of cells in prophase/prometaphase. These results demonstrate that dynein binds directly to a component of the spindle checkpoint complex through the DYNLT3 light chain. Thus, DYNLT3 contributes to dynein cargo binding specificity. These data also suggest that the subpopulation of dynein, containing the DYNLT3 light chain, may be important for chromosome congression, in addition to having a role in the transport of checkpoint proteins from the kinetochore to the spindle pole.     

A novel transforming growth factor-beta receptor-interacting protein that is also a light chain of the motor protein dynein

The phosphorylated, activated cytoplasmic domains of the transforming growth factor-beta (TGFbeta) receptors were used as probes to screen an expression library that was prepared from a highly TGFbeta-responsive intestinal epithelial cell line. One of the TGFbeta receptor-interacting proteins isolated was identified to be the mammalian homologue of the LC7 family (mLC7) of dynein light chains (DLCs). This 11-kDa cytoplasmic protein interacts with the TGFbeta receptor complex intracellularly and is phosphorylated on serine residues after ligand-receptor engagement. Forced expression of mLC7-1 induces specific TGFbeta responses, including an activation of Jun N-terminal kinase (JNK), a phosphorylation of c-Jun, and an inhibition of cell growth. Furthermore, TGFbeta induces the recruitment of mLC7-1 to the intermediate chain of dynein. A kinase-deficient form of TGFbeta RII prevents both mLC7-1 phosphorylation and interaction with the dynein intermediate chain (DIC). This is the first demonstration of a link between cytoplasmic dynein and a natural growth inhibitory cytokine. Furthermore, our results suggest that TGFbeta pathway components may use a motor protein light chain as a receptor for the recruitment and transport of specific cargo along microtublules.     

Roles of TRP14, a thioredoxin-related protein in tumor necrosis factor-alpha signaling pathways

The possible roles of a 14-kDa human thioredoxin (Trx)-related protein (TRP14) in TNF-alpha signaling were studied in comparison with those of Trx1 by RNA interference in HeLa cells. Depletion of TRP14 augmented the TNF-alpha-induced phosphorylation and degradation of I kappa B alpha as well as the consequent activation of NF-kappa B to a greater extent than did Trx1 depletion. Deficiency of TRP14 or Trx1 enhanced TNF-alpha-induced activation of caspases and subsequent apoptosis by a similar extent. The TNF-alpha-induced activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), however, was promoted by depletion of TRP14 but not by that of Trx1. Unlike Trx1, TRP14 neither associated with nor inhibited the kinase activity of apoptosis signal-regulating kinase-1 (ASK1), an upstream activator of JNK and p38. In combination with the results in the accompanying paper that TRP14 did not reduce the known substrates of Trx1, these results suggest that TRP14 modulates TNF-alpha signaling pathways, provably by interacting with proteins distinct from the targets of Trx1. In an effort to identify target proteins of TRP14, a mutant of TRP14, in which the active site cysteine (Cys(46)) was substituted with serine, was shown to form a disulfide-linked complex with LC8 cytoplasmic dynein light chain. The complex was detected in HeLa cells treated with H(2)O(2) or TNF-alpha but not in untreated cells, suggesting that LC8 cytoplasmic dynein light chain is a possible substrate of TRP14.     

M phase phosphorylation of cytoplasmic dynein intermediate chain and p150(Glued).

To understand how the dramatic cell biological changes of oocyte maturation are brought about, we have begun to identify proteins whose phosphorylation state changes during Xenopus oocyte maturation. Here we have focused on one such protein, p83. We partially purified p83, obtained peptide sequence, and identified it as the intermediate chain of cytoplasmic dynein. During oocyte maturation, dynein intermediate chain became hyperphosphorylated at the time of germinal vesicle breakdown and remained hyperphosphorylated throughout the rest of meiosis and early embryogenesis. p150(Glued), a subunit of dynactin that has been shown to bind to dynein intermediate chain, underwent similar changes in its phosphorylation. Both dynein intermediate chain and p150(Glued) also became hyperphosphorylated during M phase in XTC-2 cells and HeLa cells. Thus, two components of the dynein-dynactin complex undergo coordinated phosphorylation changes at two G2/M transitions (maturation in oocytes and mitosis in cells in culture) but remain constitutively in their M phase forms during early embryogenesis. Dynein intermediate chain and p150(Glued) phosphorylation may positively regulate mitotic processes, such as spindle assembly or orientation, or negatively regulate interphase processes such as minus-end-directed organelle trafficking.     

The African swine fever virus dynein-binding protein p54 induces infected cell apoptosis

A specific interaction of ASFV p54 protein with 8 kDa light chain cytoplasmic dynein (DLC8) has been previously characterized and this interaction is critical during virus internalization and transport to factory sites. During early phases of infection, the virus induces the initiation of apoptosis triggering activation of caspase-9 and -3. To analyze the role of the structural protein p54 in apoptosis, transient expression experiments of p54 in Vero cells were carried out which resulted in effector caspase-3 activation and apoptosis. Interestingly, p54 mutants, lacking the 13 aa dynein-binding motif lose caspase activation ability and pro-death function of p54. This is the first reported ASFV protein which induces apoptosis.     

The 8-kDa dynein light chain binds to p53-binding protein 1 and mediates DNA damage-induced p53 nuclear accumulation

The tumor suppressor protein p53 is known to undergo cytoplasmic dynein-dependent nuclear translocation in response to DNA damage. However, the molecular link between p53 and the minus end-directed microtubule motor dynein complex has not been described. We report here that the 8-kDa light chain (LC8) of dynein binds to p53-binding protein 1 (53BP1). The LC8-binding domain was mapped to a short peptide segment immediately N-terminal to the kinetochore localization region of 53BP1. The LC8-binding domain is completely separated from the p53-binding domain in 53BP1. Therefore, 53BP1 can potentially act as an adaptor to assemble p53 to the dynein complex. Unlike other known LC8-binding proteins, 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. We further showed that 53BP1 can directly associate with the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damage-induced nuclear accumulation of p53. These data illustrate that LC8 is able to function as a versatile acceptor to link a wide spectrum of molecular cargoes to the dynein motor.