Autophosphorylation-induced degradation of the Pho85 cyclin Pcl5 is essential for response to amino acid limitation.
Pho85 cyclins (Pcls), activators of the yeast cyclin-dependent kinase (CDK) Pho85, belong together with the p35 activator of mammalian CDK5 to a distinct structural cyclin class. Different Pcls target Pho85 to distinct substrates. Pcl5 targets Pho85 specifically to Gcn4, a yeast transcription factor involved in the response to amino acid starvation, eventually causing the degradation of Gcn4. Pcl5 is itself highly unstable, an instability that was postulated to be important for regulation of Gcn4 degradation. We used hybrids between different Pcls to circumscribe the substrate recognition function to the core cyclin box domain of Pcl5. Furthermore, the cyclin hybrids revealed that Pcl5 degradation is uniquely dependent on two distinct degradation signals: one N-terminal and one C-terminal to the cyclin box domain. Whereas the C-terminal degradation signal is independent of Pho85, the N-terminal degradation signal requires phosphorylation of a specific threonine residue by the Pho85 molecule bound to the cyclin. This latter mode of degradation depends on the SCF ubiquitin ligase. Degradation of Pcl5 after self-catalyzed phosphorylation ensures that activity of the Pho85/Pcl5 complex is self-limiting in vivo. We demonstrate the importance of this mechanism for the regulation of Gcn4 degradation and for cell growth under conditions of amino acid starvation.
Aviram
Sharon
S
Department of Molecular Microbiology, B. Rappaport Faculty of Medicine, Technion-I.I.T., 2 Efron St., Haifa 31096, Israel.
Simon
Einav
E
Gildor
Tsvia
T
Glaser
Fabian
F
Kornitzer
Daniel
D
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D050976:Basic-Leucine Zipper Transcription Factors; D016213:Cyclins; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; C461492:PCL5 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D013912:Threonine; D044843:SKP Cullin F-Box Protein Ligases; D018844:Cyclin-Dependent Kinases; C058605:PHO85 protein, S cerevisiae
Bioinformatics. 2005 Apr 1;21(7):951-60
15531603
Nucleic Acids Res. 1994 Dec 25;22(25):5767-8
7838736
Nature. 1995 Mar 9;374(6518):131-4
7877684
BMC Bioinformatics. 2006 Jul 12;7:339
16836757
Mol Gen Genet. 1995 Jun 10;247(5):571-8
7603436
J Biol Chem. 1999 Nov 26;274(48):33859-62
10567344
Mol Cell Biol. 1995 Feb;15(2):731-41
7823941
Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
9254694
Genes Dev. 2001 May 1;15(9):1078-92
11331604
Biochim Biophys Acta. 2004 Nov 29;1695(1-3):133-70
15571813
Nature. 1995 Jul 27;376(6538):313-20
7630397
J Bacteriol. 1973 Feb;113(2):727-38
4570606
Cell. 1985 Nov;43(1):177-88
3907851
Mol Cell Biol. 2002 Jul;22(13):4463-76
12052857
Mol Cell. 2001 Sep;8(3):657-69
11583627
Proc Natl Acad Sci U S A. 2006 Aug 29;103(35):13004-9
16916930
Mol Gen Genet. 1989 May;217(1):40-6
2671650
J Comput Chem. 2004 Oct;25(13):1605-12
15264254
J Mol Biol. 1999 Sep 17;292(2):195-202
10493868
J Biol Chem. 1998 Sep 11;273(37):24057-64
9727024
J Cell Sci. 2001 May;114(Pt 10):1811-20
11329367
Nature. 1991 Jan 10;349(6305):132-8
1846030
Yeast. 1998 Jul;14(10):953-61
9717241
Science. 1996 Mar 15;271(5255):1597-601
8599119
Nat Cell Biol. 1999 Nov;1(7):438-43
10559988
Mol Cell. 2002 May;9(5):931-43
12049731
Science. 1994 Feb 25;263(5150):1153-6
8108735
Mol Cell Biol. 1997 Mar;17(3):1212-23
9032248
Proc Natl Acad Sci U S A. 1999 Dec 7;96(25):14445-50
10588725
Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7351-5
1651502
Nature. 2003 Oct 16;425(6959):737-41
14562106
Proc Natl Acad Sci U S A. 1983 Sep;80(17):5374-8
6351059
Genes Dev. 2004 May 1;18(9):981-91
15105375
Nucleic Acids Res. 2000 Jan 1;28(1):235-42
10592235
Cell. 1983 Jun;33(2):389-96
6134587
Cell. 1993 May 7;73(3):499-511
8490963
Mol Cell. 2007 Nov 30;28(4):614-23
18042456
Proteins. 2008 Nov 1;73(2):299-319
18433058
EMBO J. 1994 Nov 15;13(22):5410-20
7957107
Front Biosci. 2000 Feb 01;5:D244-57
10704151
Bioinformatics. 2008 Jun 1;24(11):1344-8
18426805
Curr Opin Neurobiol. 2004 Jun;14(3):390-4
15194121
Eukaryot Cell. 2005 Feb;4(2):310-8
15701793
Methods Mol Biol. 2008;426:145-59
18542861
Mol Microbiol. 2007 Oct;66(2):303-14
17850263
Genes Dev. 1999 Aug 15;13(16):2039-58
10465783
Science. 1999 Apr 23;284(5414):662-5
10213692
Nature. 1999 Dec 9;402(6762):615-22
10604467
EMBO J. 1998 May 15;17(10):2759-66
9582269
Methods Enzymol. 2002;351:639-47
12073374
Mol Cell Biol. 1998 Jun;18(6):3289-99
9584169
Mol Biol Cell. 2006 Jul;17(7):2952-62
16611745
Trends Biochem Sci. 2002 Feb;27(2):87-93
11852246
Genetics. 1989 May;122(1):19-27
2659436
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10453-8
9724724
Genes Dev. 1995 Feb 15;9(4):399-409
7883165
Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2535-40
9482921
Mol Cell Biol. 2001 Jul;21(13):4347-68
11390663
Mol Biol Cell. 2000 Mar;11(3):915-27
10712509
Mol Cell Biol. 2002 Aug;22(15):5395-404
12101234
EMBO J. 1994 Dec 15;13(24):6021-30
7813440
Oncogene. 1997 Jan 30;14(4):491-8
9053846
Structure. 1995 Nov 15;3(11):1235-47
8591034
Mol Biol Cell. 1998 Sep;9(9):2393-405
9725902
Cell. 2001 Dec 14;107(6):689-701
11747804
Mol Cell Biol. 1999 Oct;19(10):7020-30
10490639
Nature. 1996 Jul 25;382(6589):325-31
8684460