Notch and Cdk5 in Zebrafish Mindbomb Mutant: Co-regulation or Coincidence?

Notch signalling is critical for the development of the nervous system. In the zebrafish mindbomb mutants, disruption of E3 ubiquitin ligase activity inhibits Notch signalling. In these mutant embryos, precocious development of primary neurons leading to depletion of neural progenitor cells results in a neurogenic phenotype characterized by defects in neural patterning and brain development. Cyclin-dependent kinase 5 (Cdk5), a predominant neuronal kinase, is involved in a variety of essential functions of the nervous system. Most recently, mammalian studies on Notch and Cdk5 regulating each other's function have been emerging. The status of Cdk5 in the mindbomb mutant embryos with excessive primary neurons is not known. In situ hybridization of the zebrafish mindbomb mutant embryos uncovered a robust upregulation in Cdk5 expression but with a reduced Cdk5 activity. The implications of these findings in both the mammalian system and zebrafish are discussed in this mini-review to provide a glimpse into the relationship between Notch and Cdk5 that may explain certain neurodevelopmental defects associated with either mutations in ubiquitin ligase or altered expression of Cdk5.

Gradual loss of DNA-PK activity from the cytoplasm is coincident with the nuclear translocation of its activator Ku during early development of Xenopus laevis.

DNA-dependent protein kinase is a serine/ threonine kinase consisting of a catalytic subunit, p460, and a regulatory subunit called Ku (p80/p70). DNA-dependent protein kinase plays a role in transcription, non-homologous recombination, and DNA repair. Previous data have shown the presence of DNA-dependent protein kinase in Xenopus oocytes and changes in its activity during vitellogenesis. Metabolic labelling studies have shown that the increased enzyme activity in vitellogenic oocytes correlates with increased levels of Ku protein, and compared to the pre- and early-vitellogenic oocytes, vitellogenic and post-vitellogenic oocytes show an increased level of DNA-dependent protein kinase activity. Whether DNA-dependent protein kinase activity is altered during early embryogenesis in Xenopus is not known. The present study demonstrates that DNA-dependent protein kinase activity is gradually lost from the cytoplasm in the early embryonic cells of blastulae and gastrulae. The gradual loss of DNA-dependent protein kinase activity during post-fertilization early embryogenesis in Xenopus is consistent with the reports on Arbacia punctulata. Immunohistochemistry of the oocytes at various stages and early embryos (gastrulae) shows that Ku70, a regulatory subunit of DNA-dependent protein kinase, is present both in the cytoplasm and nucleus in the pre-vitellogenic oocytes, full-grown post-vitellogenic oocytes and in the gastrula cells. However, Ku70 appears to accumulate in higher concentrations in the nuclei of gastrula cells. These results suggest that gradual loss of DNA-dependent protein kinase activity from the cytoplasm of the early embryos could be the consequence of Ku translocating to the nuclei that may be necessary for post-zygotic transcription followed by cellular differentiation.

Ajuba, a cytosolic LIM protein, shuttles into the nucleus and affects embryonal cell proliferation and fate decisions.

Cellular adhesive events affect cell proliferation and differentiation decisions. How cell surface events mediating adhesion transduce signals to the nucleus is not well understood. After cell-cell or cell-substratum contact, cytosolic proteins are recruited to clustered adhesion receptor complexes. One such family of cytosolic proteins found at sites of cell adhesion is the Zyxin family of LIM proteins. Here we demonstrate that the family member Ajuba was recruited to the cell surface of embryonal cells, upon aggregate formation, at sites of cell-cell contact. Ajuba contained a functional nuclear export signal and shuttled into the nucleus. Importantly, accumulation of the LIM domains of Ajuba in the nucleus of P19 embryonal cells resulted in growth inhibition and spontaneous endodermal differentiation. The differentiating effect of Ajuba mapped to the third LIM domain, whereas regulation of proliferation mapped to the first and second LIM domains. Ajuba-induced endodermal differentiation of these cells correlated with the capacity to activate c-Jun kinase and required c-Jun kinase activation. These results suggest that the cytosolic LIM protein Ajuba may provide a new mechanism to transduce signals from sites of cell adhesion to the nucleus, regulating cell growth and differentiation decisions during early development.

MEKK4 mediates differentiation in response to retinoic acid via activation of c-Jun N-terminal kinase in rat embryonal carcinoma P19 cells.

Differentiation of P19 embryonal carcinoma cells in response to the morphogen retinoic acid is regulated by Galpha(12/13) and is associated with activation of c-Jun N-terminal kinase. The role of MEKK1 and MEKK4 upstream of the c-Jun N-terminal kinase was investigated in P19 cells. P19 clones stably expressing constitutively active and dominant negative mutants of MEKK1 and MEKK4 were created and characterized. Expression of the constitutively active form of either MEKK1 or MEKK4 mimicked the action of retinoic acid, inducing these embryonal carcinoma cells to primitive endoderm. Expression of the dominant negative form of MEKK1 had no influence on the ability of retinoic acid to induce either JNK activation or primitive endoderm formation in P19 stem cells. Expression of the dominant negative form of MEKK4, in contrast, effectively blocks both morphogen-induced activation of JNK and cellular differentiation. These data identify MEKK4 as upstream of c-Jun N-terminal kinase in the pathway mediating differentiation of P19 stem cells to primitive endoderm.

Ajuba, a novel LIM protein, interacts with Grb2, augments mitogen-activated protein kinase activity in fibroblasts, and promotes meiotic maturation of Xenopus oocytes in a Grb2- and Ras-dependent manner.

LIM domain-containing proteins contribute to cell fate determination, the regulation of cell proliferation and differentiation, and remodeling of the cell cytoskeleton. These proteins can be found in the cell nucleus, cytoplasm, or both. Whether and how cytoplasmic LIM proteins contribute to the cellular response to extracellular stimuli is an area of active investigation. We have identified and characterized a new LIM protein, Ajuba. Although predominantly a cytosolic protein, in contrast to other like proteins, it did not localize to sites of cellular adhesion to extracellular matrix or interact with the actin cytoskeleton. Removal of the pre-LIM domain of Ajuba, including a putative nuclear export signal, led to an accumulation of the LIM domains in the cell nucleus. The pre-LIM domain contains two putative proline-rich SH3 recognition motifs. Ajuba specifically associated with Grb2 in vitro and in vivo. The interaction between these proteins was mediated by either SH3 domain of Grb2 and the N-terminal proline-rich pre-LIM domain of Ajuba. In fibroblasts expressing Ajuba mitogen-activated protein kinase activity persisted despite serum starvation and upon serum stimulation generated levels fivefold higher than that seen in control cells. Finally, when Ajuba was expressed in fully developed Xenopus oocytes, it promoted meiotic maturation in a Grb2- and Ras-dependent manner.

Microinjection of an antibody to the Ku protein arrests development in sea urchin embryos.

Ku is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). This enzyme plays a role in DNA repair, recombination, and transcription. It is composed of a large catalytic subunit (p460), and a regulatory heterodimer, the Ku protein, which consists of 86-kDa and 70-kDa subunits. These various components of the enzyme have been found in both eggs and embryos of the sea urchin. When variable amounts of a specific monoclonal antibody to the Ku protein (Ku 162) were injected into one cell of a 2-cell embryo of Lytechinus pictus, they caused a dose-dependent developmental arrest of the injected cell. The non-injected cell continued to develop normally. In contrast, injection of an antibody (N3H10) raised against the 70-kDa subunit of the Ku protein had no effect on development when injected into 2-cell-stage embryos. Co-injection of purified DNA-PK with the antibody reversed the antibody-mediated inhibition of development. In the fertilized egg and during the early stages of development, the DNA-PK was localized largely in the cytoplasm, but in later developmental stages, it assumed a nuclear location. On the basis of these results, we postulate that the injection of the Ku antibody either prevents the translocation of the DNA-PK into the nucleus or interferes with its enzymatic activity either in the nucleus or in the cytoplasm. In either case, the results suggest that DNA-PK plays an important role in regulating the early stages of embryogenesis in this primitive organism.

DNA-dependent protein phosphorylation activity in Xenopus is coupled to a Ku-like protein.

DNA-dependent protein kinase (DNA-PK) is a nuclear enzyme and functions as a serine/threonine kinase that has been well characterized in both the human and the mouse. The regulatory subunit of DNA-PK is the Ku autoantigen. To demonstrate that a Ku-like protein is present in Xenopus oocytes, we used immunoprecipitation analysis with a monoclonal antibody raised against human Ku antigen and autoimmune serum containing anti-Ku antibodies. Metabolic labeling studies indicate that the Ku-like protein is synthesized mainly in late vitellogenic oocytes. By using a specific peptide substrate for DNA-PK, we demonstrate the activity of a DNA-dependent protein kinase in oocyte extracts. The kinase activity requires the Ku-like protein, since extracts depleted of Ku protein by immunoadsorption with human anti-Ku antibodies fail to demonstrate the DNA-dependent phosphorylation activity. The increased enzyme activity in vitellogenic oocytes may be correlated to the increased levels of Ku protein observed in these oocytes compared to the pre- and early vitellogenic oocytes.

Autoimmunity to RNA polymerase II is focused at the carboxyl terminal domain of the large subunit.

OBJECTIVE: Previous studies have demonstrated antibodies to the large (220 kd) polypeptide subunit of RNA polymerase II (Pol II) in sera from certain patients with scleroderma. In the present study, we sought to identify the autoantigenic region on this polypeptide. METHODS: A recombinant fusion protein, corresponding to the 52-heptapeptide repeat found in the carboxyl terminal domain (CTD) of the large Pol II subunit, was used to identify 15 patient sera that contained autoantibodies. Synthetic peptides CTD7 (representing a single heptapeptide) and CTD18 (representing 2 1/2 heptapeptide repeats) were used in a competitive inhibition assay to define the specificity of these sera and the importance of the CTD as an autoantigen. RESULTS: All 15 sera immunoprecipitated the Pol II subunit from radiolabeled cell extracts, and 11 of them bound the CTD fusion protein in immunoblots. Immunoprecipitation of Pol II was completely inhibited by CTD18 in 5 sera and partially inhibited in 4 additional sera. CONCLUSION: These results indicate that the CTD heptapeptide repeat is a focal point for autoimmune responses in scleroderma. It is likely that the repetitive sequence and high content of charged residues of this structure contribute to its role as an autoantigen.

Fundulus heteroclitus vitellogenin: the deduced primary structure of a piscine precursor to noncrystalline, liquid-phase yolk protein.

We have cloned and sequenced a cDNA encoding a vitellogenin (Vtg) from the mummichog, Fundulus heteroclitus, an estuarine teleost. We constructed a liver cDNA library against RNA from estrogen-treated male mummichogs. Five overlapping cDNA clones totalling 5,197 bp were isolated through a combination of degenerate oligonucleotide probing of the library and PCR. The cDNA sequence contains a 5,112 bp open reading frame. The predicted primary structure of the deduced 1,704-amino-acid protein is 30-40% identical to other documented chordate Vtgs, establishing this Vtg as a member of the ancient Vtg gene family. Of the previously reported chordate Vtg sequences (Xenopus laevis, Gallus domesticus, Ichthyomyzon unicuspis, and Acipenser transmontanus), all four act as precursor proteins to a yolk which is eventually rendered insoluble under physiological conditions, either as crystalline platelets or as noncrystalline granules. The yolk of F. heteroclitus, on the other hand, remains in a soluble state throughout oocyte growth. The putative F. heteroclitus Vtg contains a polyserine region with a relative serine composition that is 10-20% higher than that observed for the other Vtgs. The trinucleotide repeats encoding the characteristic polyserine tracts of the phosvitin region follow a previously reported trend: TCX codons on the 5' end and AGY codons toward the 3' end. Whether the difference in Vtg primary structure between F. heteroclitus and that of other chordates is responsible for the differences in yolk structure remains to be elucidated. As the first complete teleost Vtg to be reported, these data will aid in designing nucleotide and immunological probes for detecting Vtg as a reproductive status indicator in F. heteroclitus and other piscine species.

Expression of extracellular ligand-binding domain of murine guanylate cyclase/atrial natriuretic factor receptor cDNA in Escherichia coli.

The membrane-bound form of guanylate cyclase/atrial natriuretic factor receptor (GC/ANF-R) is a 135 kDa transmembrane glycoprotein which binds ANF with high affinity. We have expressed the extracellular ligand-binding domain of murine guanylate cyclase ANF-R (GC/ANFR-LBD) cDNA in Escherichia coli. The cDNA encoding the extracellular ANF-binding domain (nucleotide positions covering from 432-1755 base pair) of GC/ANF-R was amplified by polymerase chain reaction, cloned into BamHI site of pGEX-3X prokaryotic expression vector and was transfected into E. coli, strain JM101. After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of bacterial cells, the GC/ANFR-LBD was expressed as the glutathione-S-transferase (GST) fusion protein, yielding a molecular mass of 70 kDa. The expressed fusion protein was characterized for binding affinity to both full length and truncated ANF molecules. After expression in E. coli, the binding of 125I-ANF to the extracellular region of GC/ANF-R was similar and corresponded to the pharmacological class of native receptor protein. The 70 kDa fusion product was purified as a predominant single protein band by glutathione-affinity chromatography. These findings establish that E. coli may be utilized as an effective heterologous model system to delineate the structure-function analysis of guanylate cyclase-coupled ANF receptor molecules.

Oogenesis in Fundulus heteroclitus. VI. Establishment and verification of conditions for vitellogenin incorporation by oocytes in vitro.

A procedure was developed for studying vitellogenin (VTG) incorporation by vitellogenic oocytes of Fundulus heteroclitus in vitro. Since homologous VTG can be obtained from this animal only with great difficulty, the use of [32P]VTG from Xenopus laevis was explored as an alternative. Vitellogenic as well as maturational-stage oocytes were found to sequester X. laevis [32P]VTG from the medium, and incorporation was found to be linear with time for at least up to 12 hr. Once incorporated into the oocyte, [32P]VTG did not appear to undergo turnover. The effect of different [32P]VTG concentrations on incorporation indicated that the uptake mechanism was saturable. Unlabeled F. heteroclitus VTG and X. laevis VTG were also found to compete effectively with X. laevis [32P]VTG, whereas bovine serum albumin did not. These results represent the first documentation of a successful culture system for receptor-mediated VTG incorporation by teleost oocytes.