PubMed · 8252620
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T Hunter. 1993-12-03. Braking the cycle.. https://doi.org/10.1016/0092-8674(93)90528-x
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Levels of cyclins and cyclin-dependent kinase (Cdk) inhibitors are tightly controlled during normal cell proliferation and are frequently dysregulated in cancerous cells. In melanoma, cyclin D1 is highly expressed and downregulation of the Cdk inhibitor, p27(Kip1), is associated with a poor prognosis. Mutant B-RAF is frequently expressed in melanoma and overrides growth factor and matrix adhesion control of cyclin D1 and p27(Kip1) levels in human melanocytes. Here, we demonstrate that p27(Kip1) expression is regulated by multiple mechanisms in melanoma cells. B-RAF regulates p27(Kip1) mRNA abundance independently of cyclin D1. Additionally, B-RAF and cyclin D1 control the levels of S-phase kinase-associated protein 2 (Skp2) that directs ubiquitin-mediated proteolysis of p27(Kip1). The cofactor for Skp2, Cdc kinase subunit 1 (Cks1) controls levels of Skp2 in melanoma cells and acts jointly with Skp2 to regulate p27(Kip1) levels. Importantly, expression of Cks1 is regulated by B-RAF and cyclin D1 at the mRNA level. Reduced Cks1 or Skp2 expression and enhanced p27(Kip1) levels inhibit melanoma cell growth. In summary, p27(Kip1) expression in melanoma is regulated by B-RAF at the mRNA level, and via B-RAF and cyclin D1 control of Cks1/Skp2-mediated proteolysis.
Skp2 is the substrate recognition subunit of the multi-subunit ubiquitin ligase SCF(Skp2). It consists of an N-terminal F-box domain that binds to the Skp1 subunit and thereby tethers it to the SCF catalytic core, and an elongated C-terminal domain comprising ten Leucine-rich repeats (LRR) that binds the substrate. A small accessory protein, Cks1, is required for SCF(Skp2) to target certain substrates, including the Cyclin-dependent kinase inhibitor p27. Here we have used hydrogen/deuterium exchange monitored by mass spectrometry to investigate the mode of action of Cks1 on SCF(Skp2). We show that complex formation between Cks1 and Skp2 causes conformational changes in both proteins in regions distant from the respective binding sites. We find that Skp2 interacts with a localised region of Cks1 but the interaction causes a global change in the hydrogen exchange behaviour of Cks1. Also, whilst Cks1 binds to the most C-terminal LRRs of the elongated Skp2 molecule, the interaction induces conformational changes at the distant N-terminal LRRs, close to the F-box motif. Further, binding of Cks1 to Skp2 significantly stabilises the interaction between Skp2 and Skp1. The results reveal that the C-terminal substrate recognition region of Skp2 is coupled to the N-terminal Skp1-binding region and thereby to the SCF catalytic core; this result adds to the model proposed previously that, whilst the principal function of the F-box protein is to recruit the substrate, an additional function may be to help position the substrate in an optimal way within the SCF complex to enable efficient ubiquitin transfer.
The ability of the human fungal pathogen Candida albicans to transit its cell shape is important for its pathogenicity. To obtain additional evidence that the cell cycle of C. albicans is associated with its morphology, we generated and characterized a conditional mutant of C. albicans CDC28, a cyclin-dependent kinase. In the constructed strain, the expression of CDC28 was regulated by the MET3 promoter and could be repressed in the presence of methionine and cysteine. Cdc28p-depleted cells demonstrated highly polarized growth and wider filaments than serum-induced hyphae. Hyphae-specific genes, HWP1, RBT4 and ECE1, were activated in the elongated filaments caused by the Cdc28p depletion. Furthermore, the protein expression levels of the transcription factors involved in morphological transition, Efg1p, Nrg1p, Rbf1p, Rim101p, Fkh2p and Tec1p, decreased under conditions that repress CDC28 expression. Taken together, these data indicate that repression of CDC28 affected the protein levels of the morphology-related transcription factors, the regulation of hyphae-specific genes and cell shape in C. albicans.