Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase

Kristi Levine; Lee Kiang; Matthew D. Jacobson; Robert P. Fisher; Frederick R. Cross

doi:10.1016/S1097-2765(00)80337-8

Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase

Kristi Levine, Lee Kiang, Matthew D. Jacobson, Robert P. Fisher, Frederick R. Cross

Research output: Contribution to journal › Article › peer-review

12 Scopus citations

Abstract

To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.

Original language	English
Pages (from-to)	353-363
Number of pages	11
Journal	Molecular Cell
Volume	4
Issue number	3
DOIs	https://doi.org/10.1016/S1097-2765(00)80337-8
State	Published - Sep 1999
Externally published	Yes

Access to Document

10.1016/S1097-2765(00)80337-8

Cite this

@article{fcc62d51a8be4ebfa00e2df74dd0c07b,

title = "Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase",

abstract = "To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.",

author = "Kristi Levine and Lee Kiang and Jacobson, {Matthew D.} and Fisher, {Robert P.} and Cross, {Frederick R.}",

note = "Funding Information: We thank Nicola Pavletich for useful discussions and Ray Deshaies for plasmids. This work was supported by PHS grants GM47238 and GM58459. K. L. was an HHMI predoctoral fellow. ",

year = "1999",

month = sep,

doi = "10.1016/S1097-2765(00)80337-8",

language = "English",

volume = "4",

pages = "353--363",

journal = "Molecular Cell",

issn = "1097-2765",

publisher = "Cell Press",

number = "3",

}

TY - JOUR

T1 - Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase

AU - Levine, Kristi

AU - Kiang, Lee

AU - Jacobson, Matthew D.

AU - Fisher, Robert P.

AU - Cross, Frederick R.

N1 - Funding Information: We thank Nicola Pavletich for useful discussions and Ray Deshaies for plasmids. This work was supported by PHS grants GM47238 and GM58459. K. L. was an HHMI predoctoral fellow.

PY - 1999/9

Y1 - 1999/9

N2 - To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.

AB - To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.

UR - http://www.scopus.com/inward/record.url?scp=0033197840&partnerID=8YFLogxK

U2 - 10.1016/S1097-2765(00)80337-8

DO - 10.1016/S1097-2765(00)80337-8

M3 - Article

C2 - 10518216

AN - SCOPUS:0033197840

SN - 1097-2765

VL - 4

SP - 353

EP - 363

JO - Molecular Cell

JF - Molecular Cell

IS - 3

ER -

Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase

Abstract

Access to Document

Fingerprint

Cite this