F-Box Protein Specificity for G1 Cyclins Is Dictated by Subcellular Localization

Levels of G1 cyclins fluctuate in response to environmental cues and couple mitotic signaling to cell cycle entry. The G1 cyclin Cln3 is a key regulator of cell size and cell cycle entry in budding yeast. Cln3 degradation is essential for proper cell cycle control; however, the mechanisms that control Cln3 degradation are largely unknown. Here we show that two SCF ubiquitin ligases, SCFCdc4 and SCFGrr1, redundantly target Cln3 for degradation. While the F-box proteins (FBPs) Cdc4 and Grr1 were previously thought to target non-overlapping sets of substrates, we find that Cdc4 and Grr1 each bind to all 3 G1 cyclins in cell extracts, yet only Cln3 is redundantly targeted in vivo, due in part to its nuclear localization. The related cyclin Cln2 is cytoplasmic and exclusively targeted by Grr1. However, Cdc4 can interact with Cdk-phosphorylated Cln2 and target it for degradation when cytoplasmic Cdc4 localization is forced in vivo. These findings suggest that Cdc4 and Grr1 may share additional redundant targets and, consistent with this possibility, grr1Δ cdc4-1 cells demonstrate a CLN3-independent synergistic growth defect. Our findings demonstrate that structurally distinct FBPs are capable of interacting with some of the same substrates; however, in vivo specificity is achieved in part by subcellular localization. Additionally, the FBPs Cdc4 and Grr1 are partially redundant for proliferation and viability, likely sharing additional redundant substrates whose degradation is important for cell cycle progression.

[1]  B. Futcher,et al.  The Cln3‐Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation. , 1992, The EMBO journal.

[2]  R. Deshaies,et al.  SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities. , 1997, Molecular biology of the cell.

[3]  Y. Xiong,et al.  ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity. , 1999, Molecular cell.

[4]  S. Reed,et al.  Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover , 2003, Nature Reviews Molecular Cell Biology.

[5]  Mike Tyers,et al.  CDK Activity Antagonizes Whi5, an Inhibitor of G1/S Transcription in Yeast , 2004, Cell.

[6]  A. Klein-Szanto,et al.  Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex. , 2006, Molecular cell.

[7]  Ronald W. Davis,et al.  A genome-wide transcriptional analysis of the mitotic cell cycle. , 1998, Molecular cell.

[8]  Michael Ruogu Zhang,et al.  Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. , 1998, Molecular biology of the cell.

[9]  M. Hochstrasser,et al.  SUMO-independent in vivo activity of a SUMO-targeted ubiquitin ligase toward a short-lived transcription factor. , 2010, Genes & development.

[10]  E. O’Shea,et al.  Quantification of protein half-lives in the budding yeast proteome , 2006, Proceedings of the National Academy of Sciences.

[11]  M. Tyers,et al.  Genome-wide surveys for phosphorylation-dependent substrates of SCF ubiquitin ligases. , 2005, Methods in enzymology.

[12]  J Wade Harper,et al.  Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. , 2007, Molecular cell.

[13]  蟹江 共春.  Genetic reevaluation of the role of F-box proteins in cyclin D1 degradation , 2012 .

[14]  Mike Tyers,et al.  A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin. , 2004, Biochimica et biophysica acta.

[15]  David O. Morgan,et al.  Positive feedback sharpens the anaphase switch , 2008, Nature.

[16]  J. Gervais,et al.  Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[17]  E. Garí,et al.  Cyclin Cln3 is retained at the ER and released by the J chaperone Ydj1 in late G1 to trigger cell cycle entry. , 2007, Molecular cell.

[18]  A. Klein-Szanto,et al.  Phosphorylation-Dependent Ubiquitination of Cyclin D1 by the SCFFBX4-αB Crystallin Complex , 2006 .

[19]  M. Pagano,et al.  SnapShot: F Box Proteins II , 2009, Cell.

[20]  Mike Tyers,et al.  F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex , 1997, Cell.

[21]  R. Deshaies,et al.  A Complex of Cdc4p, Skp1p, and Cdc53p/Cullin Catalyzes Ubiquitination of the Phosphorylated CDK Inhibitor Sic1p , 1997, Cell.

[22]  I. Herskowitz,et al.  Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast. , 1997, Genes & development.

[23]  C Mann,et al.  G1 cyclin turnover and nutrient uptake are controlled by a common pathway in yeast. , 1995, Genes & development.

[24]  Frank McCormick,et al.  A Critical Role for FBXW8 and MAPK in Cyclin D1 Degradation and Cancer Cell Proliferation , 2006, PloS one.

[25]  F. Cross,et al.  DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae , 1988, Molecular and cellular biology.

[26]  B. Futcher,et al.  p34Cdc28-mediated control of Cln3 cyclin degradation , 1995, Molecular and cellular biology.

[27]  Pei-Jung Lu,et al.  A Novel Mechanism by Which Thiazolidinediones Facilitate the Proteasomal Degradation of Cyclin D1 in Cancer Cells* , 2008, Journal of Biological Chemistry.

[28]  Curt Wittenberg,et al.  Cln3 Activates G1-Specific Transcription via Phosphorylation of the SBF Bound Repressor Whi5 , 2004, Cell.

[29]  K. Shokat,et al.  Targets of the cyclin-dependent kinase Cdk1 , 2003, Nature.

[30]  Frederick R. Cross,et al.  Mechanisms Controlling Subcellular Localization of the G1 Cyclins Cln2p and Cln3p in Budding Yeast , 2001, Molecular and Cellular Biology.

[31]  C. Berset,et al.  Transferable Domain in the G1 Cyclin Cln2 Sufficient To Switch Degradation of Sic1 from the E3 Ubiquitin Ligase SCFCdc4 to SCFGrr1 , 2002, Molecular and Cellular Biology.

[32]  S. Elledge,et al.  Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1. , 1999, Science.

[33]  C. Wittenberg,et al.  Rapid Degradation of the G1 Cyclin Cln2 Induced by CDK-Dependent Phosphorylation , 1996, Science.

[34]  David O. Morgan,et al.  Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase , 2011, Nature.

[35]  Stephen J. Elledge,et al.  SKP1 Connects Cell Cycle Regulators to the Ubiquitin Proteolysis Machinery through a Novel Motif, the F-Box , 1996, Cell.

[36]  M. Dai,et al.  Balance of Yin and Yang: ubiquitylation-mediated regulation of p53 and c-Myc. , 2006, Neoplasia.

[37]  F. Yamao,et al.  An essential function of Grr1 for the degradation of Cln2 is to act as a binding core that links Cln2 to Skp1. , 1998, Journal of cell science.

[38]  B. Andrews,et al.  Systematic mapping of genetic interaction networks. , 2009, Annual review of genetics.

[39]  S. Gygi,et al.  Global Analysis of Cdk1 Substrate Phosphorylation Sites Provides Insights into Evolution , 2009, Science.

[40]  L. Drury,et al.  The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast , 1997, The EMBO journal.

[41]  Frederick R. Cross,et al.  Distinct Subcellular Localization Patterns Contribute to Functional Specificity of the Cln2 and Cln3 Cyclins of Saccharomyces cerevisiae , 2000, Molecular and Cellular Biology.

[42]  K Nasmyth,et al.  Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. , 1999, Genes & development.

[43]  B. Futcher,et al.  Relationship between the function and the location of G1 cyclins in S. cerevisiae. , 2001, Journal of cell science.

[44]  F. Cross,et al.  The yeast Cln3 protein is an unstable activator of Cdc28 , 1993, Molecular and cellular biology.

[45]  David P. Toczyski,et al.  A proteomic screen reveals SCFGrr1 targets that regulate the glycolytic–gluconeogenic switch , 2007, Nature Cell Biology.

[46]  G. Tokiwa,et al.  The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. , 1988, The EMBO journal.

[47]  William Stafford Noble,et al.  The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle. , 2006, Genes & development.

[48]  M. Pagano,et al.  SnapShot: F Box Proteins I , 2009, Cell.

[49]  E. Kipreos,et al.  Multiple degradation pathways regulate versatile CIP/KIP CDK inhibitors. , 2012, Trends in cell biology.

[50]  C Longaretti,et al.  Nuclear‐specific degradation of Far1 is controlled by the localization of the F‐box protein Cdc4 , 2000, The EMBO journal.

[51]  Frederick R. Cross,et al.  Positive feedback of G1 cyclins ensures coherent cell cycle entry , 2008, Nature.

[52]  Nicholas A. Lyons,et al.  Cdk1-dependent destruction of Eco1 prevents cohesion establishment after S phase. , 2011, Molecular cell.