Tài liệu Báo cáo Y học: CK2btes gene encodes a testis-specific isoform of the regulatory subunit of

CK2btes gene encodes a testis-specific isoform of the regulatory

subunit of casein kinase 2 in Drosophila melanogaster

Alla I. Kalmykova1

, Yuri Y. Shevelyov1

, Oksana O. Polesskaya1,

*, Anna A. Dobritsa1,

†,

Alexandra G. Evstafieva2

, Brigitte Boldyreff3

, Olaf-Georg Issinger3 and Vladimir A. Gvozdev1

1

Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia; 2

Belozersky Institute of Physico-Chemical Biology,

Center of Molecular Medicine, Moscow State University, Russia; 3

Department of Biochemistry and Molecular Biology,

University of Southern Denmark, Odense, Denmark

An earlier described CK2btes gene of Drosophila melano￾gasteris shown to encode a male germline specific isoform of

regulatory b subunit of casein kinase 2. Western-analysis

using anti-CK2btes Ig revealed CK2btes protein in

Drosophila testes extract. Expression of a CK2btes–

b-galactosidase fusion protein driven by the CK2btes pro￾moter was found in transgenic flies at postmitotic stages of

spermatogenesis. Examination of biochemical characteris￾tics of a recombinant CK2btes protein expressed in

Escherichia colirevealed properties similar to those of CK2b:

(a) CK2btes protein stimulates CK2a catalytic activity

toward synthetic peptide; (b) it inhibits phosphorylation of

calmodulin and mediates stimulation of CK2a by polylysine;

(c) it is able to form (CK2btes)2 dimers, as well as

(CK2a)2(CK2btes)2 tetramers. Using the yeast two-hybrid

system and coimmunoprecipitation analysis of protein

extract from Drosophila testes, we demonstrated an associ￾ation between CK2btes and CK2a. Northern-analysis has

shown that another regulatory (b¢) subunit found recently in

D. melanogaster genome is also testis-specific. Thus, we

describe the first example of two tissue-specific regulatory

subunits of CK2 which might serve to provide CK2 sub￾strate recognition during spermatogenesis.

Keywords: spermatogenesis; casein kinase 2; CK2 bsubunit;

CK2btes; testes.

Protein kinase CK2 is involved in such general cell processes

as cell cycle regulation, transcriptional control, signal

transduction, development and proliferation [1–4]. More

than 160 different proteins serve as substrates for CK2.

Phosphorylation by CK2 has been found to affect activity

of such Drosophila proteins pivotal for realization of early

developmental program, as Cut-homeodeomain protein,

Cactus and Antennapedia [5–7]. A CK2 holoenzyme

consists of two a- (or a¢-) and two b subunits. The a subunit

of CK2 possesses catalytic activity and the regulatory

b subunit was shown to enhance stability of the holoenzyme,

activate CK2a and provide substrate specificity and CK2

targeting in cells. In spite of CK2b being ubiquitously

represented among eukaryotes, it is far less conserved in

comparison with the catalytic CK2a. This fact might be

explained by a wide spectrum of substrates and partner

proteins interacting with CK2b as a regulatory subunit.

Moreover, other functions, besides being a part of the CK2

holoenzyme, can be ascribed to the b subunit. For example,

it has been demonstrated that the CK2 b subunit is involved

in the regulation of catalytic activity of two other protein

kinases (A-raf and Mos kinases [8–10]). The conclusion that

CK2b has a more general functions is supported by the fact

that significant imbalance of its amount in respect to

a subunit is found in tumor cells and some mammalian

tissues such as testicles [11, 12].

Recently it was shown that the CK2 activity as well as the

CK2 protein level are mostly elevated in rat and mouse

testicles [12, 13]. An important role for the CK2 activity in

spermatogenesis was clearly shown by a Ôknock-outÕ of the

CK2a¢ gene in mice resulting in a male sterile phenotype

[14]. Spermatogenesis is a complex differentiation process

comprising mitotic and meiotic divisions of germline stem

cells followed by sperm morphogenesis. This process is

known to have a lot of common features in Drosophila and

mammals [15]. However, genetic control and molecular

mechanisms of both Drosophila and mammalian sperma￾togenesis are still poorly understood.

The genomes of most eukaryotes including mammals

carry a single gene encoding b subunit of CK2. Only

Saccharomyces cerevisiae and Arabidopsis thaliana are

known to have two and three isoforms of CK2b, respect￾ively [16, 17]. Recently, we have described in Drosophila the

SSL gene [18], later renamed CK2btes [19], as a first

candidate on the role of a tissue-specific isoform of the CK2

regulatory subunit. This gene is expressed exclusively in

testes and encodes a protein sharing 45% amino-acid

identity with the ubiquitous Drosophila b subunit. Another

potential Drosophila tissue-specific CK2 regulatory subunit

(b¢) was identified in the yeast two-hybrid screen where

Correspondence to Y. Y. Shevelyov, Department of Molecular

Genetics of Animals, Institute of Molecular Genetics, 123182,

Kurchatov Sq. 2, Moscow, Russia. Fax: + 7 095 1960221;

Tel.: + 7 095 1961909; E-mail: [email protected]

Abbreviations: CK2, casein kinase 2; CK2b, CK2 b subunit; CK2a,

CK2 a subunit; IP, immunoprecipitation; RNAi, RNA interference;

dsRNA, double stranded RNA; X-gal, 5-bromo-4-chloro-3-indolyl

b-galactopyranoside.

*Present address: Molecular Neurobiology Branch, NIDA, NIH, 5500

Nathan Shock Drive, Baltimore, MD, 21224, USA.

 Present address: Department of Molecular, Cellular and Develop￾mental Biology, Yale University, New Haven, CT 06520, USA.

(Received 25 September 2001, revised 7 December 2001, accepted 14

January 2002)

Eur. J. Biochem. 269, 1418–1427 (2002) Ó FEBS 2002