Rkp1/Cpc2, a Fission Yeast rACK1 Homolog, Is Involved in Actin Cytoskeleton Organization through Protein Kinase C, Pck2, Signaling

doi:10.1006/bbrc.2001.4535

Biochemical and Biophysical Research Communications

Volume 282, Issue 1, 23 March 2001, Pages 10-15

https://doi.org/10.1006/bbrc.2001.4535 Get rights and content

Abstract

The Rkp1/Cpc2, a fission yeast RACK1 homolog, interacted with Pck2, one of the known PKC homologs, in vivo and in vitro. The rkp1-deletion mutants (Δrkp1) are elongated and the pck2-deletion mutant (Δpck2) showed abnormal morphology. The double-deletion mutant (Δrkp1Δpck2) showed more aberrant cell shapes and was sensitive to high salt concentration. Both Δrkp1 and Δpck2 cells were sensitive to latrunculin B (Lat B) which inhibits actin polymerization. The cells expressing the human RACK1 homolog complemented the latrunculin B sensitivity of Δrkp1 indicating that human RACK1 is a functional homolog of Rkp1/Cpc2. We propose that Rkp1/Cpc2 may function as a receptor for Pck2 in the regulation of actin cytoskeleton organization during cell wall synthesis and morphogenesis of Schizosaccharomyces pombe.

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A total of 437 human full-length cDNAs isolated by microarray analysis of liver and/or gastric cancer tissues were evaluated for their relevance to cancer using the fission yeast Schizosaccharomyces pombe. Overexpression of 161 human cDNAs in S. pombe caused growth inhibition and/or morphological changes, which can be considered as cancer-related phenotypes of S. pombe. Sixteen genes causing growth defects and morphological changes at the same time were chosen to validate their ostensible oncogenic properties. They were highly expressed in liver and/or gastric cancer cell lines. Also, when the mouse embryonic fibroblast cell type NIH3T3 was transfected with these genes, the proliferation rates of cells were increased by 32% to 120%. This study demonstrates that fission yeast can be used as an advantageous and powerful tool for the rapid screening of human genes relevant to cancer. Furthermore, the human genes screened can be tested further as diagnostic markers and potential therapeutic targets for liver and stomach cancers. They also can be studied further for the elucidation of mechanisms involved in carcinogenesis.
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The receptor for activated C kinase 1 (RACK1) is a conserved scaffold protein that helps regulate a range of cell activities including cell growth, shape, and protein translation. We report that a homologue of RACK1 is required for cytokinesis in pathogenic Trypanosoma brucei. The protein, referred to as TRACK, is comprised of WD repeat elements and can complement cpc2 null mutants of Schizosaccharomyces pombe. TRACK is expressed throughout the trypanosome life cycle and is distributed predominantly in a perinuclear region and the cytoplasm but not along the endoplasmic reticulum, mitochondrion, or cleavage furrow of dividing cells. When tetracycline-inducible RNA interference (RNAi) is used to deplete the cellular content of TRACK, the cells remain metabolically active, but growth is inhibited. In bloodstream forms, growth arrest is due to a delay in the onset of cytokinesis. By contrast, procyclic forms are able to initiate cytokinesis in the absence of TRACK but arrest midway through cell cleavage. The RNAi cells undergo multiple rounds of partial cytokinesis and accumulate nuclei and cytoplasmic extensions with attached flagella. The TRACK RNAi construct is also inducible within infected mice. Under these conditions parasites are eliminated from peripheral blood within 3 days post-infection. Taken as a whole, these data indicate that trypanosomes utilize a RACK1 homologue to regulate the final stages of mitosis. Moreover, disrupting the interaction between TRACK and its partners might be targeted in the design of novel therapies.
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Receptors for activated C kinases (RACKs) are scaffold proteins that anchor diverse signaling proteins and are involved in modulating cell cycle. We report the cloning and cellular localization of a RACK ortholog (PfRACK) in the human malaria parasite Plasmodium falciparum. The full-length transcript obtained by 3^′ and 5^′ RACE has 1.4 kbp with a predicted ORF of 972 bp, coding for a protein with 323 residues of 35.8 kDa molecular weight and pI 6.38. PfRACK has 59% and 60% identity at the amino acid level to Chlamydomonas reinhardtii and Danio rerio RACKs, respectively, presenting seven WD40 motifs and retaining the conserved domains in repeats III (DVFSVSF) and VI (STINSLCF) that are important for PKC binding. Semi-quantitative RT-PCR revealed that PfRACK is constitutively expressed in the intraerythrocytic stages of P. falciparum. Using confocal microscopy, PfRACK was immunolocalized in all parasite stages, being conspicuously spread throughout the schizont. The high similarity of PfRACK to those previously described in other organisms, as well as its constitutive expression in Plasmodium asexual stages, suggests that it might play a key role in the regulatory processes of malaria parasite life cycle.
Pleckstrin homology domain interacts with Rkp1/Cpc2, a RACK1 homolog, to modulate Pck2-mediated signaling process in Schizosaccharomyces pombe
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Rkp1/Cpc2, a fission yeast RACK1 homolog, interacts with Pck2, a PKC homolog, and is involved in the regulation of pck2-mediated signaling process. The N-terminal region of split pleckstrin homology domain (nPH) in human PLC-γ1 bound to Rkp1/Cpc2 concomitantly with Pck2. nPH inhibited kinase activity of GST-Pck2 purified from Schizosaccharomyces pombe in vitro. The lethality induced by pck2⁺ overexpression was suppressed by coexpression of either rkp1⁺ or nPH domain. This result suggests that Rkp1/Cpc2 interacts with PH domain-containing protein and regulates the Pck2-mediated signaling process in S. pombe.
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^☆: Abbreviations used: PKC, protein kinase C; RACK, receptor for activated protein kinase C; MBP, maltose binding protein; GST, glutathione S-transferase; GFP, green fluorescence protein of jelly fish Aequorea victoria.

¹: To whom correspondence and reprint requests should be addressed. Fax: 82-42-860-4409. E-mail: [email protected].

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Biochemical and Biophysical Research Communications

Abstract

References (23)

MAP kinase pathways in yeast: For mating and more

Cell

The proliferation of MAP kinase signaling pathways in yeast

Curr. Opin. Cell Biol.

Activation of yeast protein kinase C by Rho1 GTPase

J. Biol. Chem.

Characterization of Pak2p, a pleckstrin homology domain-containing, p21-activated protein kinase from fission yeast

J. Biol. Chem.

The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform

J. Biol. Chem.

Two novel protein kinase C-related genes of fission yeast are essential for cell viability and implicated in cell shape control

EMBO J.

Schizosaccharomyces pombe protein kinase C homologues, pck1p and pck2p, are targets of rho1p and rho2p and differentially regulate cell integrity

J. Cell Sci.

Localisation of the Schizosaccharomyces pombe rho1p GTPase and its involvement in the organisation of the actin cytoskeleton

J. Cell Sci.

Rho1p, a yeast protein at the interface between cell polarization and morphogenesis

Science

Cloning of an intracellular receptor for protein kinase C: A homolog of the beta subunit of G proteins

Proc. Natl. Acad. Sci. USA

C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo

J. Biol. Chem.

Cited by (19)

Rapid screen of human genes for relevance to cancer using fission yeast

The RACK1 homologue from Trypanosoma brucei is required for the onset and progression of cytokinesis

Human malaria parasites display a receptor for activated C kinase ortholog

Pleckstrin homology domain interacts with Rkp1/Cpc2, a RACK1 homolog, to modulate Pck2-mediated signaling process in Schizosaccharomyces pombe

A WD40 Protein Encoding Gene Fvcpc2 Positively Regulates Mushroom Development and Yield in Flammulina velutipes

RACK(1) to the future - A historical perspective

Regular ArticleRkp1/Cpc2, a Fission Yeast rACK1 Homolog, Is Involved in Actin Cytoskeleton Organization through Protein Kinase C, Pck2, Signaling☆

Abstract

Cell

Curr. Opin. Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Two novel protein kinase C-related genes of fission yeast are essential for cell viability and implicated in cell shape control

EMBO J.

Schizosaccharomyces pombe protein kinase C homologues, pck1p and pck2p, are targets of rho1p and rho2p and differentially regulate cell integrity

J. Cell Sci.

Localisation of the Schizosaccharomyces pombe rho1p GTPase and its involvement in the organisation of the actin cytoskeleton

J. Cell Sci.

Rho1p, a yeast protein at the interface between cell polarization and morphogenesis

Science

Cloning of an intracellular receptor for protein kinase C: A homolog of the beta subunit of G proteins

Proc. Natl. Acad. Sci. USA

C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo

J. Biol. Chem.

Regular Article
Rkp1/Cpc2, a Fission Yeast rACK1 Homolog, Is Involved in Actin Cytoskeleton Organization through Protein Kinase C, Pck2, Signaling☆