SUMMARYSmoking is strongly associated with abnormalities in histone-to-protamine transition and with alteration of protamine expression in human spermatozoa. A proper protamine to histone ratio is, however, essential for sperm chromatin maturity and DNA integrity. Alterations in these sperm nuclear proteins were observed in infertile men. The present prospective study is aimed at evaluating the possible relationship among smoking, semen quality and the histone-to-protamine transition ratio in mature spermatozoa. Histone H2B and protamine 1 (P1) and 2 (P2) were quantified using acid-urea polyacrylamide gel electrophoresis in the spermatozoa of 35 smokers and 19 non-smokers. Levels of lipid peroxidation marker malondialdehyde (MDA) were measured in seminal plasma by thiobarbituric acid assay. Cotinine concentrations were determined in seminal plasma using an enzyme-linked immunosorbent assay. Histone H2B levels in smokers (292.27 AE 58.24 ng/10 6 ) were significantly higher (p = 0.001) than that of non-smokers (109.1 AE 43.70 ng/10 6 ), besides, a significant difference (p > 0.0001) was found for the P1 and P2 ratio between smokers (1.71 AE 0.071) and non-smokers (1.05 AE 0.033). The H2B/(H2B+P1 + P2) ratio (0.29 AE 0.71) of smokers were significantly higher (p = <0.0001) than that of non-smokers (0.12 AE 0.01). The concentrations of MDA (lM) (7.13 AE 1.15) and cotinine (ng/mL) (60.44 AE 31.32) in seminal plasma of smokers were significantly higher (p = 0.001) than those in the samples of the non-smoker group (4.42 AE 1.16 and 2.01 AE 2.84 respectively). In addition, smokers showed significantly (p ≤ 0.002) lower sperm count, motility (p = 0.018), vitality (p = 0.009) and membrane integrity (p = 0.0001) than non-smokers. These results reveal that patients who smoke possess a higher proportion of spermatozoa with an alteration of the histone to protamine ratio than patients who do not smoke, and suggest that cigarette smoking may inversely affect male fertility.

Cyclin dependent kinases are regulated by phosphorylation and dephosphorylation of the catalytic cdk subunits, by assembly with specific cyclins and by specific inhibitor molecules. Recently, it turned out that cyclins are also phosphoproteins, which means that they are also potential targets for a regulation by phosphorylation and dephosphorylation. Here, we show that cyclin H was phosphorylated by protein kinase CK2. Like most other CK2 substrates cyclin H was much better phosphorylated by the CK2 holoenzyme than by the a-subunit alone. By using point mutants derived from the cyclin H sequence we mapped the CK2 phosphorylation site at threonine 315 at the C-terminal end of cyclin H. Phosphorylation at this position had no influence on the assembly of the cyclin H/cdk7/Mat1 complex. However, phosphorylation at amino acid 315 of cyclin H turned out to be critical for a full cyclin H/cdk7/Mat1 kinase activity when the CTD peptide of RNA polymerase II or cdk2 was used as a substrate.

Mdm2 is a cellular oncoprotein the most obvious function of which is the down-regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro. Computer analysis of the primary amino acid sequence of mdm2 revealed 19 putative CK2 phosphorylation sites. By using deletion mutants of mdm2 and a peptide library we identified the serine residue at position 269 which lies within a canonical CK2 consensus sequence (EGQELSDEDDE) as the most important CK2 phosphorylation site. Moreover, by using the mdm2 S269A mutant for in vitro phosphorylation assays this site was shown to be phosphorylated by CK2. Binding studies revealed that phosphorylation of mdm2 at S269 does not have any influence on the binding of p53 to mdm2.Keywords: mdm2; oncogene; p53; phosphorylation site; protein kinase CK2.Mdm2 (mouse double minute 2) was first identified as a gene responsible for the spontaneous transformation of a mouse 3T3 cell line [1,2]. This cell line contains 25±30 copies of paired chromatin bodies called double minutes which express three genes, mdm1, 2 and 3. As the mdm2 gene mediates transformation in several tissue culture systems [2,3] and was found to be overexpressed in many different human tumours [4±6] it was classified as an oncogene. At least seven different transcripts have been identified [2,7,8] in mouse and human cell lines; the functions of these have still to be elucidated. The largest human transcript is translated into a protein of 491 amino acids which migrates in SDS/PAGE at an apparent molecular mass of < 90 kDa [9]. The most prominent function of mdm2 is the down-regulation of the transactivating functions of the growth suppressor p53 [10]. The proteins are closely connected by an autoregulatory feedback loop [11]: mdm2 is transactivated by p53 [12] and binds p53, probably only in its tetrameric form [13], with an N-terminal region between amino acids 19 and 102 [14] thus blocking directly the transactivation function of p53. Moreover, it has been shown that mdm2 binds to p53 in the nucleus and transports it to a cytoplasmic proteasome where p53 is degraded by a ubiquitin-mediated pathway [15]. It has even been suggested that mdm2 acts as a ubiquitin ligase for p53 in this degradation process [16]. Thus, mdm2 antagonizes the growth suppressing functions of p53 in at least two ways.Mdm2 stimulates cell proliferation not only by antagonizing the transactivation function of p53 but also by blocking the growth suppressor Rb [17] and thus, possibly enhancing the transcriptional activity of E2F/DP1 [18]. Furthermore, mdm2 has been shown to bind TAF II 250 which positively correlates with the activation of the cyclin A promoter [19]. By interacting with different components of the transcription/ translation mach...

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory β-subunit and the catalytic α- or α'-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2β and TNP1 were found in early spermatogenesis. CK2α, CK2α', TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2β was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.

CK2 is a serine/threonine protein kinase, which is so important for many aspects of cellular regulation that life without CK2 is impossible. Here, we analysed CK2 during adipogenic differentiation of human mesenchymal stem cells (hMSCs). With progress of the differentiation CK2 protein level and the kinase activity decreased. Whereas CK2α remained in the nucleus during differentiation, the localization of CK2β showed a dynamic shuttling in the course of differentiation. Over the last years a large number of inhibitors of CK2 kinase activity were generated with the idea to use them in cancer therapy. Our results show that two highly specific inhibitors of CK2, CX-4945 and quinalizarin, reduced its kinase activity in proliferating hMSC with a similar efficiency. CK2 inhibition by quinalizarin resulted in nearly complete inhibition of differentiation whereas, in the presence of CX-4945, differentiation proceeded similar to the controls. In this case, differentiation was accompanied by the loss of CX-4945 inhibitory function. By analysing the subcellular localization of PPARγ2, we found a shift from a nuclear localization at the beginning of differentiation to a more cytoplasmic localization in the presence of quinalizarin. Our data further show for the first time that a certain level of CK2 kinase activity is required for adipogenic stem cell differentiation and that inhibition of CK2 resulted in an altered localization of PPARγ2, an early regulator of differentiation.