Erich A. Nigg

In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. Phosphorylation of lamin proteins is believed to cause lamina disassembly during meiotic and mitotic M phase, but the M phase-specific lamin kinase has not been identified. Here we show that the cdc2 kinase, a major element implicated in controlling the eukaryotic cell cycle, phosphorylates chicken B-type lamins in vitro on sites that are specifically phosphorylated during M phase in vivo. Concomitantly, cdc2 kinase is capable of inducing lamina depolymerization upon incubation with isolated nuclei. One of the target sites of cdc2 kinase is identified as a motif (SPTR) conserved in the N-terminal domain of all lamin proteins. These results lead us to propose that mitotic disassembly of the nuclear lamina results from direct phosphorylation of lamins by cdc2 kinase.

Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase

Following the identification of the cdc2 kinase as a major element controlling entry of cells into mitosis, it is important to define the physiological target range of this enzyme. Here, we demonstrate that two major nucleolar proteins, nucleolin and NO38, are highly phosphorylated during mitosis. Importantly, the two nucleolar proteins are also phosphorylated by highly purified starfish cdc2 kinase in vitro, on sites that correspond to those observed specifically during mitosis in vivo. A repeated motif (TPXKK) is identified as the likely mitotic phosphoacceptor site in nucleolin, in that a synthetic peptide mimicking this site functions as both a substrate and a competitive inhibitor of cdc2 kinase. These results identify two novel candidate substrates for cdc2 kinase, and they implicate protein phosphorylation in controlling mitotic changes in nucleolar structure and activity.

Cellular substrates of p34cdc2 and its companion cyclin-dependent kinases

Cyclin-dependent kinase (cdks) are key components of the engine that drives the cell proliferation cycle in all eukaryotes. These kinases are related to p34(cdc2) and associate with regulatory subunits belonging to the cyclin family. To understand how cdks promote cell cycle progression, it will be important to identify their physiological substrates and to determine how phosphorylation influences the functions of these substrates. This article discusses recent progress as well as some of the problems related to the quest for cdk substrates.

Nuclear export of proteins : the role of nuclear retention

Proteins that shuttle between nucleus and cytoplasm are implicated in transport and signal transduction processes. Using assays based on interspecies heterokaryons and microinjection of Xenopus oocytes, we examined what structural features determine nuclear export of shuttling proteins. Three classes of proteins were studied: first, wild-type and mutant forms of nucleolin, one of the first shuttling proteins identified; second, artificial nuclear reporter proteins derived from cytoplasmic pyruvate kinase; and third, wild-type and mutant lamins differing in their abilities to be incorporated into the lamina. Our results show that a protein does not require positively acting export signals to be transported from nucleus to cytoplasm; instead, its shuttling ability is limited primarily by intranuclear interactions. We conclude that nucleocytoplasmic shuttling is a general phenomenon not restricted to proteins involved in nucleocytoplasmic transport.

Mechanisms of signal transduction to the cell nucleus

Publisher Summary This chapter outlines the early consequences of surface receptor stimulation, and summarizes the information about the structure of nuclear pores and the transport of macromolecules across the nuclear envelope. It is assumed that the signal transduction between cytoplasm and nucleus involves primarily the translocation of proteins between the two compartments. According to the classic two-step model of steroid hormone action, the receptor activation is accompanied by translocation of ligand–receptor complexes from cytoplasm to nucleus. The steroid-free receptors form the macromolecular complexes through association with a 90-kDa heat shock protein, and hormone-dependent release from this complex may be required for receptor function. Protein phosphorylation is known to represent a major mechanism for controlling the nuclear activities and gene transcription. Immunocytochemical studies suggest that nuclear effects of cAMP are mediated by the C subunit. In response to the cAMP elevation, the catalytic subunit of protein kinases (PK-A) translocates from a perinuclear location to the nucleus.

Cell Cycle: The NIMA kinase joins forces with Cdc2

The NIMA and Cdc2 protein kinases cooperate to regulate mitosis in Aspergillus nidulans. NIMA-related pathways have now begun to emerge in higher eukaryotes.

Structure and developmental expression of chicken nucleolin and NO38: coordinate expression of two abundant non-ribosomal nucleolar proteins.

We report the complete primary structures of two major chicken non-ribosomal nucleolar proteins known as nucleolin/C23 and NO38/B23, respectively. By comparison with homologous proteins from other species, this sequence information contributes to the identification of evolutionarily conserved motifs that may be relevant to the function and subcellular distribution of the two proteins. Using cDNA probes and monoclonal antibodies, we have also studied the expression of nucleolin and NO38 in the course of chicken embryogenesis. In all tissues examined, Northern analyses revealed single hybridization signals for nucleolin (at 3.0 kb) and NO38 (at 1.6 kb), and no evidence was obtained for multiple protein products. In total embryos between days 3 and 11 after egg laying, nucleolin and NO38 mRNA and protein levels decreased in parallel (2-5-fold), suggesting transcriptional down-regulation of expression. Coordinate expression of nucleolin and NO38 was observed also when examining individual tissues at various stages of development. Interestingly, however, there was no consistent correlation between relative mRNA and protein levels. In particular, several adult tissues contained exceedingly low levels of either nucleolin or NO38, despite the presence of large amounts of corresponding mRNAs. From these results we conclude, first, that the expression of nucleolin and NO38 is controlled coordinately, and, second, that regulation is likely to involve both transcriptional and posttranscriptional mechanisms.