Abstract. We report here that disruption of a recently discovered kinesin-like protein in Drosophila melanogaster, KLP61F, results in a mitotic mutation lethal to the organism. We show that in the absence of KLP61F function, spindle poles fail to separate, resulting in the formation of monopolar mitotic spindles. The resulting phenotype of metaphase arrest with polyploid cells is reminiscent of that seen in the fungal bimC and cut7 mutations, where it has also been shown that spindle pole bodies are not segregated. KLP61F is specifically expressed in proliferating tissues during embryonic and larval development, consistent with a primary role in cell division. The structural and functional homology of the KLP61F, bimC, cut7, and Eg5 kinesin-like proteins demonstrates the existence of a conserved family of kinesin-like molecules important for spindle pole separation and mitotic spindle dynamics.T HE existence of microtubule-dependent force generating molecules has been known for nearly thirty years (reviewed in Vallee and Shpetner, 1990). The intrinsic polarity of the microtubule suggests there should be two classes of molecules capable of transducing force in either direction along the fiber. In general, dyneins move organelles along microtubules in the minus-end direction, whereas kinesins have been implicated in plus end-directed movement (reviewed in Endow, 1991;Goldstein, 1991; Mclntosh and Pfarr, 1991;Sawin and Scholey, 1991;Vallee, 1991). The matriarch of the kinesin superfamily (kinesin) was discovered in squid axoplasm and as such, is likely to function in axonal transport (Allen et al., 1985;Brady, 1985;Vale et al., 1985). As expected for this role, mutation of the kinesin heavy chain in Drosophila melanogaster results in lethality with associated disruption of neuromuscular function (Gho et al., 1992;Saxton et al., 1991).Since the initial identification of the kinesin heavy chain, a number of studies have led to the conclusion that a superfamily of kinesin-like proteins (KLPs) ~ plays diverse roles in cellular functions in all single-and multi-cellular eukaryotes examined to date (reviewed in Endow, 1991;Goldstein, 1991). These KLPs all share homology within the motor domain of the protein which is involved in ATP hydrolysis, microtubule binding, and force generation. Two PCR-based screens (using primers to conserved sequences within the mechanochemical region) in Drosophila melanogaster have identified six, and probably more, genes encoding potential KLPs (Endow and Hatsumi, 1991;Stewart et al., 1991). Functional analysis is incomplete at best, and awaits the discovery of mutations in these putative KLP genes.In addition to axonal transport, what other cellular processes may require microtubule-based motility? In the cell, the minus-ends of microtubules are embedded in the centrosome or microtubule organizing center, while the plus ends extend into the cytoplasm. The most dramatic cellular rearrangements occur during cell division. A mitotic spindle is first constructed from the duplicated centrosome...
Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK␣ and -, which bind to a basic docking motif in the C terminus of MK2 and which subsequently phosphorylate its regulatory sites. As a result of activation MK2 is exported from the nucleus to the cytoplasm and cotransports active p38 MAPK to this compartment. Here we show that the amount of p38 MAPK is significantly reduced in cells and tissues lacking MK2, indicating a stabilizing effect of MK2 for p38. Using a murine knockout model, we have previously shown that elimination of MK2 leads to a dramatic reduction of tumor necrosis factor (TNF) production in response to lipopolysaccharide. To further elucidate the role of MK2 in p38 MAPK stabilization and in TNF biosynthesis, we analyzed the ability of two MK2 isoforms and several MK2 mutants to restore both p38 MAPK protein levels and TNF biosynthesis in macrophages. We show that MK2 stabilizes p38 MAPK through its C terminus and that MK2 catalytic activity does not contribute to this stabilization. Importantly, we demonstrate that stabilizing p38 MAPK does not restore TNF biosynthesis. TNF biosynthesis is only restored with MK2 catalytic activity. We further show that, in MK2-deficient macrophages, formation of filopodia in response to extracellular stimuli is reduced. In addition, migration of MK2-deficient mouse embryonic fibroblasts (MEFs) and smooth muscle cells on fibronectin is dramatically reduced. Interestingly, reintroducing catalytic MK2 activity into MEFs alone is not sufficient to revert the migratory phenotype of these cells. In addition to catalytic activity, the proline-rich N-terminal region is necessary for rescuing the migratory phenotype. These data indicate that catalytic activity of MK2 is required for both cytokine production and cell migration. However, the proline-rich MK2 N terminus provides a distinct role restricted to cell migration.The stress-activated kinase mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is a direct substrate of p38 MAPK␣ and - (also designated stress-activated protein kinase 2a and 2b). Phosphorylation of MK2 by p38 MAPK serves a dual function. First, it results in the activation of MK2 kinase activity, which in turn leads to the phosphorylation of substrates of MK2 such as small heat shock protein Hsp25/27, tyrosine hydroxylase, and leukocyte-specific protein 1. In addition, MK2 determines the subcellular localization of p38 MAPK. Phosphorylation of MK2 by p38 MAPK unmasks a nuclear export signal (NES) in the C-terminal part of the molecule (4). This unmasking is a prerequisite for nucleocytoplasmic transport of MK2, which in turn coexports activated p38 MAPK from the nucleus to the cytoplasm. Notably, MK2 catalytic activity is not required for cotransport of p38 (1). Using a targeted deletion of MK2, we have shown that MK2 is posttranscriptionally required for the lipopolysaccharide (LPS)-induced production of several cytokines including tumor necrosis factor (TNF) and interleuki...
Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mutant, tsNY72-4, express a set of pp60v-src-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, we have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transformation per se. Significantly, however, v-src produced prolonged, and in some cases kinetically complex, patterns of induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) induced mRNAs with kinetics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was repressed by TPA at 1 hr, after which this mRNA was permanently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, contains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.
We isolated a cDNA for p20K, a secreted protein preferentially synthesized in nonproliferating cells. p20K mRNA and protein levels declined rapidly following treatment with various mitogens. DNA sequence analysis of the p20K cDNA predicted a novel protein distantly related to alpha 2 mu-globulin and plasma retinol-binding protein.
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