Tài liệu Báo cáo khoa học: Protein kinase CK2 activates the atypical Rio1p kinase and promotes its

Protein kinase CK2 activates the atypical Rio1p kinase

and promotes its cell-cycle phase-dependent degradation

in yeast

Michaela Angermayr1,*, Elisabeth Hochleitner2,†, Friedrich Lottspeich2 and Wolfhard Bandlow1

1 Department Biologie I, Bereich Genetik, Ludwig-Maximilians-Universita¨t Mu¨nchen, Germany

2 Max-Planck-Institut fu¨r Biochemie, Martinsried, Germany

The protein kinase casein kinase 2 (CK2) is ubiquitous

in eukaryotes and is responsible for the Ser⁄Thr phos￾phorylation of a large number of protein substrates

[1–3]. The active holoenzyme is most often a hetero￾tetramer composed of two catalytic a subunits, a

(encoded by CKA1) and a¢ (encoded by CKA2), and

two regulatory b subunits, b and b¢ in Saccharomyces

cerevisiae (CKB1 and CKB2). The enzyme occurs in all

possible combinations of a and b subunits [4,5]. In

yeast, deletion of the gene for one of the two catalytic

subunits has little effect, but deletion of both homolo￾gous genes results in loss of viability [6]. To date, more

than 300 endogenous CK2 substrates are known to be

involved in quite diverse processes, e.g. cell proliferation,

signal transduction, transcriptional regulation, transla￾tion and metabolism [3]. Despite this eminent role in

strictly regulated cellular processes and although CK2

is indispensable for cell life, CK2 activity by itself is

apparently unregulated [4,5], although some fluctua￾tion in activity in correlation with cell-cycle progres￾sion has been seen in cultured mammalian cells [7,8].

The physiological effect of substrate phosphorylation

is surprisingly low with almost all targets described to

date [9,10]. In S. cerevisiae, it has been shown using

temperature-sensitive CKA2 alleles that protein kinase

CK2 is required for passage across the G2 ⁄M bound￾ary and for cell-cycle progression through the G1

phase [11].

Keywords

atypical protein kinase; casein kinase 2

substrate; cell-cycle phase-dependent

degradation; protein–protein interactions;

2Rio1 protein kinase

1Correspondence

M. Angermayr

E-mail: [email protected]

Present address

*ac-Pharma AG, Oberhaching, Germany

†Wacker Chemie AG, Burghausen, Germany

(Received 16 May 2007, revised 11 July

2007, accepted 16 July 2007)

doi:10.1111/j.1742-4658.2007.05993.x

Using co-immunoprecipitation combined with MS analysis, we identified

the a¢ subunit of casein kinase 2 (CK2) as an interaction partner of the

atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2

from Dcka1 or Dcka2 mutant extracts shows that Rio1p preferentially

interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential

and sufficient for this interaction. Six C-terminally located clustered serines

were identified as the only CK2 sites present in Rio1p. Replacement of all

six serine residues by aspartate, mimicking constitutive phosphorylation,

stimulates Rio1p kinase activity about twofold in vitro compared with

wild-type or the corresponding (S > A)6 mutant proteins. Both mutant

alleles (S > A)6 or (S > D)6 complement in vivo, however, growth of the

RIO1 (S > A)6 mutant is greatly retarded and shows a cell-cycle pheno￾type, whereas the behaviour of the RIO1 (S > D)6 mutant is indistinguish￾able from wild-type. This suggests that phosphorylation by protein kinase

CK2 leads to moderate activation of Rio1p in vivo and promotes cell pro￾liferation. Physiological studies indicate that phosphorylation by CK2 ren￾ders the Rio1 protein kinase susceptible to proteolytic degradation at the

G1 ⁄ S transition in the cell-division cycle, whereas the non-phosphorylated

version is resistant.

Abbreviations

Aky2p, adenylate kinase 2; CK2, casein kinase 2; GST, glutathione S-transferase.

4654 FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS