Tài liệu Báo cáo khoa học: Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell-cycle phase-dependent degradation in yeast pdf

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 Department Biologie I, Bereich Genetik, Ludwig-Maximilians-Universitat Munchen, Germany ă ă Max-Planck-Institut fur Biochemie, Martinsried, Germany ă Keywords atypical protein kinase; casein kinase substrate; cell-cycle phase-dependent degradation; protein–protein interactions; Rio1 protein kinase Correspondence M Angermayr E-mail: M.Angermayr@lrz.uni-muenchen.de 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 (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 phenotype, whereas the behaviour of the RIO1 (S > D)6 mutant is indistinguishable from wild-type This suggests that phosphorylation by protein kinase CK2 leads to moderate activation of Rio1p in vivo and promotes cell proliferation Physiological studies indicate that phosphorylation by CK2 renders 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 The protein kinase casein kinase (CK2) is ubiquitous in eukaryotes and is responsible for the Ser ⁄ Thr phosphorylation of a large number of protein substrates [1–3] The active holoenzyme is most often a heterotetramer 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 homologous 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, translation 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 fluctuation in activity in correlation with cell-cycle progression 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 boundary and for cell-cycle progression through the G1 phase [11] 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 M Angermayr et al Rio1p from yeast has been identified as the founding member of a novel family of atypical protein serine kinases [12–14] It is essential in yeast and is only distantly related to previously characterized protein kinases The RIO1 gene is transcribed constitutively at an extremely low level [15,16], and Rio1p is a very low abundant protein [12] Yeast has only one such gene, whereas at least two orthologues of Rio1p are present in higher eukaryotes The primary structure of the catalytic domain and the N-terminal so-called Rio1-family domain are highly conserved from archaea to man, whereas a great deal of sequence variation resides in the extreme N- and C-terminal portions [13] We found that Rio1p plays a role in cell-cycle progression Yeast cells deprived of RIO1 lose minichromosomes at an increased rate relative to wild-type and arrest either as large G1 cells (i.e late in the G1 phase of the cell-division cycle) or as large-budded M cells with a single DNA mass at the bud neck and short spindles This indicates that Rio1p is simultaneously required in the G1 phase and for the onset of anaphase (and ⁄ or nuclear division and chromosome segregation) [12] Vanrobays et al [17] obtained evidence from a synthetic lethal screen with GAR1, an essential gene required for 18S rRNA maturation, that Rio1p might be involved in ribosome biogenesis However, it is feasible that Rio1p has more than one target and plays a role in several pathways in yeast (as may be deduced from the fact that two orthologues occur in higher eukaryotes) [13] Rio1 protein kinase is regulated by CK2 The biological role of Rio1p or even the pathways in which the Rio1 protein kinase is involved are far from being understood Targets or interaction partners have not been identified as yet We report here first attempts to identify interaction partners and found that the activity and cellular concentration of Rio1p are regulated by phosphorylation through CK2 in a cell-cycle-dependent fashion Results Rio1p interacts with Cka2p In an effort to identify the interaction partners of the essential Rio1p kinase, we performed co-purification experiments after overexpression of an N-terminally myc3-tagged version of Rio1p from yeast extracts Subsequently, proteins were identified by mass spectrometry We found several presumptive Rio1p interaction partners which co-purified exclusively with full-length Rio1p but not with a C-terminally truncated version (M Angermayr, unpublished) Among them we identified Cka2p, one of the two a subunits of protein kinase CK2 using this approach, however, we did not recover the other a subunit (Cka1p) or any of the b subunits (Ckb1p or Ckb2p) To verify interactions between Rio1p and Cka2p, RIO1 was fused, at its 5¢-end, to a myc3 tag-encoding sequence (the RIO1 deletion mutants used are compiled in Fig 1), and CKA2 was equipped, at its 5¢-end, with a nucleotide sequence encoding an HA3-tag, both Fig Rio1 protein kinase (A) Domains of the Rio1p kinase are indicated (according to the classification of Hanks et al [45]); candidates for CK2 phosphorylation are indicated by an asterisk (*); pos., positions relative to the translational start codon A, N-Terminal domain; B, Rio1 family-specific domain (R-domain); CK, serine-rich acidic domain (pos 402–435, CK2 domain); and K, lysine-rich C-terminal domain (K domain, pos 436–484) (B) N- and C-terminally truncated versions of the Rio1 protein used in this study; amino acid sequences expressed are indicated (C) CK2 phosphorylation sites in the C-terminal domain (CK2 domain) of Rio1p; the respective S residues are emphasized and positions are indicated (pos 402–435); the respective CK2 phosphorylation sites are numbered consecutively (1–6) FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4655 Rio1 protein kinase is regulated by CK2 M Angermayr et al transcribed from the inducible GAL10 promoter Using yeast extracts, co-immunoprecipitations were performed once with anti-(myc agarose) to purifiy Rio1p and once with HA antibodies to purifiy Cka2p Co-purified Cka2p or Rio1p was subsequently detected by western analysis with HA or myc antibodies, respectively (Fig 2) Because we presumed that the C-terminal portion of Rio1p was involved in protein–protein inter- Fig Co-immunopurification experiments and identification of domains essential for Rio1p–Cka2p interactions (A) HA3-tagged Cka2p was immunoprecipitated using HA antibodies and protein A–Sepharose; co-purified myc3-tagged Rio1p was subsequently detected by immunodetection with myc antibodies (B) Control detection of total immunoprecipitated HA3-tagged Cka2p using HA antibodies (C) Myc3-tagged Rio1 proteins were immunoprecipitated using anti-(myc agarose) Co-purified HA3-tagged Cka2p was subsequently detected by immunodecoration with HA antibodies (D) Control detection of total immunoprecipitated myc3-tagged Rio1 proteins using myc antibodies (E) Myc3-tagged Rio1 proteins were immunoprecipitated with anti-(myc agarose) Co-purified Cka2p was subsequently detected by HA antibodies in a western blot (F) Immunoprecipitation efficiencies of the Rio1 proteins were controlled by immunodetection with myc antibodies Cka2p, HA3-Cka2p which was still immunoreactive during the second detection on the same blot in (F) p1, p2, control plasmids containing exclusively the GAL10 promoter and the myc3- or HA3-tag, respectively fl, fulllength Rio1p; 1–408p, C-terminally truncated Rio1p 4656 actions and might serve as a substrate for CK2, we also used a C-terminally truncated version of Rio1p, 1–408 [12] as a control (Fig 1B) In addition, we investigated whether a catalytically inactive allele of RIO1, Rio1D244N [12] interacts with HA3–Cka2p as well (inactive RIO1 alleles were rescued by the, untagged, genomic copy of RIO1) When Cka2p was immunoprecipitated with HA antibodies, we detected the active or inactive versions of Rio1p in a subsequent western blot by using myc antibodies, indicating that both active and inactive Rio1 proteins interact with Cka2p (Fig 2A,B) This was also true, when anti-(myc agarose) was applied to precipitate Rio1p (Fig 2C,D) The interaction of Rio1p with CK2 is extremely stable and resistant to extensive washing (not shown), whereas the C-terminally truncated Rio1p (1–408) displays only weak interactions with Cka2p (Fig 2A,C; in C, only a faint signal was detected) The above results indicated that the C-terminus of Rio1p might play a role in the interaction with Cka2p in yeast cells To determine domains of Rio1p which are necessary and sufficient to interact with Cka2p, we produced a series of RIO1 truncations Products containing only amino acids 1–46 or 1–76 turned out to be unstable in yeast In order to test the possible importance of the N–terminus in the interaction with CK2 we could therefore use only N-terminal truncations (46–484, 76–484; Fig 1) in this experiment We made another C-terminal truncation (1–402; in this construct an additional presumptive CK2 phosphorylation site has been deleted in comparison with 1–408) In a complementary experiment, we used a construct containing exclusively the C-terminal part of Rio1p (starting immediately C-terminal adjacent to domain XI, amino acids 335–484) Co-purification was performed using anti-(myc agarose), and co-purified HA3–Cka2p was subsequently identified by western blot analysis using HA antibodies (Fig 2E,F) Interactions between Rio1p and Cka2p were abolished completely as soon as the 82 C-terminal amino acids of Rio1p were deleted (constructs 1–402, 46–402 or 76–402) Conversely, Cka2p co-purified with the C-terminal fragment of Rio1p (amino acids 335– 484) Thus, the C-terminus is necessary and sufficient to establish this interaction N)Terminal truncations behave like full-length Rio1p in this respect and not affect Rio1p–Cka2p complex formation, demonstrating that the N-terminus of Rio1p has no bearing on the interactions between Rio1p and Cka2p Rio1p is a target of CK2 The above results indicated that Rio1p and Cka2p interact with one another and that the interaction FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS M Angermayr et al Rio1 protein kinase is regulated by CK2 Fig Rio1p is a target of CK2 (A) Affinity-purified recombinant inactive GST-tagged Rio1 proteins were incubated with (+) or without (–) recombinant CK2 in the presence of [32P]ATP[cP], electrophoresed and autoradiographed (B) Coomassie Brilliant Blue-staining of the respective gel GST served as a negative control in in vitro kinase assays *, degradation product of the recombinant full-length version of Rio1p (present in lanes and 3) (C) Different recombinant GST-tagged Rio1 protein full-length or truncated versions were affinity-purified, incubated with (+) or without (–) recombinant CK2 in the presence of [32P]ATP[cP], and detected by autoradiography after SDS ⁄ PAGE * denotes degradation products of full-length Rio1p and the C-terminal portion (amino acids 335–484) (D) Coomassie Brilliant Blue-stained gel of (C) domain might involve the C-terminal segment of Rio1p To analyse whether Rio1p and Cka2p interact directly, i.e without bridging factors from yeast, we purified recombinant glutathione S-transferase (GST)– Rio1p from Escherichia coli Rio1 wild-type protein has autophosphorylation activity, but GST-fused Rio1p is enzymatically inactive, as observed with several kinases Therefore, this fusion protein is a suitable substrate to unambiguously test whether Rio1p is a target of CK2 GST–Rio1p was incubated without and with recombinant human CK2 holoenzyme in the presence of [32P]ATP[cP] (Fig 3A) GST served as an additional negative control in the kinase assays No autophosphorylation (absence of CK2) was detected corroborating that GST–Rio1p is inactive GST–Rio1p phosphorylation signals were detected only after incubation with CK2 C-Terminally truncated GST–Rio1p (1–408p) was poorly phosphorylated by CK2 when the signal strengths of precipitated GST–Rio1p and GST1–408p were compared (Coomassie Brilliant Bluestained gel, Fig 3B) No phosphate incorporation was detected when amino acids 1–402 of Rio1p served as a substrate for CK2 (Fig 3C), suggesting the absence of CK2 sites N-terminal of position 402 in the catalytic domain and the presence of several phosphorylation sites for CK2 in the C-terminal portion of Rio1p, one (or more) of them in the segment between positions 402 and 408 In the complementary experiment, the C-terminal fragment of Rio1p (335–484p) was heavily phosphorylated (Fig 3C) These results show that: (a) Rio1p and CK2 interact directly in vitro, because both proteins are of recombinant origin; (b) recombinant CK2 holoenzyme has the capacity to phosphorylate Rio1p; and (c) the CK2 phosphorylation sites of Rio1p lie within a region between amino acid 402 and the C-terminus at position 484 To provide evidence that Rio1p is also a target of CK2 in vivo, we examined the extent of Rio1p phosphorylation from extracts of a Dcka1 or Dcka2 yeast mutant, respectively As controls we used an inactive allele of RIO1, Rio1-D244N, and the truncated Rio1(1– 402-D244N) mutant, the latter is both inactive and not phosphorylated by Cka2p (Fig 4) Using yeast extracts obtained from cells wild-type for CKA1 and CKA2, tagged versions of Rio1p or Rio1-D244Np were heavily phosphorylated, whereas only a weak signal was detected with Rio1(1–402-D244N)p (Fig 4A) (This residual phosphorylation is independent of both Rio1p and CK2 kinase activities and, likely attributable to the action of still another protein kinase; M Angermayr, unpublished) However, in the Dcka1 or Dcka2 genetic backgrounds, phosphate incorporation dropped to  40% (Dcka1) or 25% (Dcka2) with either Rio1p or Rio1(D244N)p (Fig 4C) indicating that Rio1p is phosphorylated mainly by a heterotetramer containing both FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4657 Rio1 protein kinase is regulated by CK2 M Angermayr et al Fig Phosphorylation of Rio1p by CK2 after Co-purification from yeast cellular extracts (A) Myc3-tagged Rio1 proteins were immunoprecipitated with anti-(myc agarose) using yeast extracts from wild-type-, Dcka1-, and Dcka2 yeast strains; immunoprecipitates were incubated in the presence of [32P]ATP[cP], and detected by autoradiography after SDS ⁄ PAGE fl, full-length Rio1p (B) Coomassie Brilliant Blue-stained gel; IgG, antibody heavy chain; fl, fulllength proteins (C) Quantitative evaluation of phosphate incorporation into the respective Rio1 proteins with respect to the different genetic backgrounds (wild-type, Dcka1, Dcka2) Cka1p and Cka2p less efficiently by a heterotetramer composed of two Cka2p subunits, and only poorly by the two Cka1p catalytic subunits-comprising holoenzyme The observed differences in phosphate incorporation ( 20%) between Rio1 wild-type and mutant (D244N) proteins are likely attributable to simultaneous autophosphorylation of Rio1p and ⁄ or the action of still another protein kinase (M Angermayr, unpublished observations) The above results indicate that Rio1p–CK2 interactions are not restricted to Cka2p, but might be exerted via Cka1p as well To test the capacity of tagged versions of Cka1p or Cka2p to compete with the respective residual version of CKA in either a Dcka1 or Dcka2 genetic background, we performed co-immunoprecipitation experiments using an HA3tagged version of Cka1p (Fig 5) Cka1p interacts with Rio1p, although to a much lesser extent than Cka2p 4658 Fig Interaction of Rio1p with Cka1p or Cka2p (A) Myc3-Rio1p [myc3-RIO1] was immunoprecipitated from yeast extracts from different genetic backgrounds (wild-type, Dcka1, Dcka2), coexpressing either HA3-Cka1p, [HA3-CKA1], or HA3-Cka2p, [HA3-CKA2], respectively Co-purified HA3-Cka1p or HA3-Cka2p was subsequently identified by immunodetection in a western blot using HA antibodies IgG, antibody heavy chain (B) Control of immunoprecipitation efficiencies of myc3-Rio1p by western blot (C) Quantitative evaluation (normalized to the strongest signal in 5B) of relative interaction efficiencies between Rio1p and Cka1p or Rio1p and Cka2p, respectively, in yeast strains disrupted for either CKA1 or CKA2 Genotypes are indicated below (C), and alleles in brackets denote the tagged (and immunoprecipitated) isozyme of CK2 (Quantitative evaluation is only shown for the respective Dcka1 or Dcka2 genetic background, respectively) Quantification of co-immunoprecipitates in the Dcka1 or Dcka2 genetic background showed that Rio1p binds with higher affinity to Cka2p, corroborating the results obtained with in vitro phosphorylation experiments Protein kinase CK2 phosphorylates six clustered serine residues of Rio1p Computational analyses (http://scansite.mit.edu/ motifscan_seq.phtml) indicated that several (four to six, depending on the stringency set) high- and low-affinity FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS M Angermayr et al phosphorylation sites for CK2 might exist exclusively in the C-terminal part of Rio1p To determine whether these sites are functional, we changed the candidate Ser residues one by one to Ala using site-directed in vitro mutagenesis In vitro kinase assays with recombinant CK2 holoenzyme and the respective (enzymatically inactive) recombinant GST-fused Rio1 mutant proteins as substrates revealed a total of six tightly clustered serine residues as CK2 phosphorylation sites [S402 (S1), S403 (S2), S409 (S3), S416 (S4), S417 (S5), S419 (S6)] consecutively numbered 1–6; cf Fig 1C (Fig 6) The total number of CK2 phosphorylation sites of Rio1p was deduced from experiments with several single, double, and triple S to A mutations; not all combinations are shown Recombinant (inactive) GST-fused RIO1 mutant alleles in which all six presumptive phosphorylation sites for CK2 had been mutated exhibited no residual phosphorylation signal at all after incubation with recombinant CK2 proving that all CK2 recognition sites within the Rio1p kinase had been destroyed Rio1 protein kinase is regulated by CK2 Quantitative evaluation of phosphate incorporation indicated that CK2 displays different affinities towards the respective serine residues (Fig 6C) Phosphorylation by CK2 stimulates Rio1p kinase activity To investigate the possible physiological importance of Rio1p phosphorylation by CK2, we changed all six CK2 phosphorylation sites from S to either A (S > A)6, or D (S > D)6, respectively N-Terminally His6-tagged wild-type and mutant proteins were purified from E coli and analysed in vitro by kinase assays using histone H2B [12] as a substrate (Fig 7) Quantification revealed comparable rates of phosphate incorporation into the heterologous substrate by the Rio1 wild-type and (S > A)6 mutant proteins, as expected for recombinant proteins lacking modifications (e.g unphosphorylated by CK2) However, when the CK2 sites were changed to D (S > D)6 (mimicking permanently CK2-phosphorylated Rio1p), Rio1 mutant protein kinase activity was stimulated approximately twofold To prove the functionality of the respective CK2 phosphorylation sites in yeast, we incubated immunoprecipitated myc3-tagged Rio1 wild-type, (S > A)6, or (S > D)6 mutant proteins from yeast in the presence of [32P]ATP[cP] (Fig 8) Quantification of phosphate incorporation into the respective Rio1p versions showed that the Rio1 wild-type protein was heavily phosphorylated (Fig 8C) However, when the six CK2 phosphorylation sites were mutated to either alanine or aspartate, phosphate incorporation dropped to  20 or 40%, respectively, which reflects autophosphorylation of Rio1p and ⁄ or the presence of a site for another as yet unidentified kinase which co-immunoprecipitated together with Rio1p in addition to CK2 Biological implications of phosphorylation of Rio1p by CK2 Fig Mutational analyses of the CK2-sites of Rio1p (A) Recombinant GST-fused Rio1 mutant proteins were incubated without (–) or with (+) recombinant CK2 in the presence of [32P]ATP[cP], separated by SDS ⁄ PAGE and autoradiographed (B) Coomassie Brilliant Blue-stained gel (C) Quantification of phosphate incorporation (relative to GST–Rio1p precipitated amounts) into the respective Rio1p mutant proteins by protein kinase CK2; * denotes degradation products In order to examine the possible biological importance of Rio1p phosphorylation in vivo, we tested whether substitution of all six CK2 phosphorylation sites in Rio1p by either A or D (mimicking unphosphorylated or permanently phosphorylated Rio1p, respectively) has any consequences on yeast viability or growth rate For this purpose the respective mutant alleles were brought into the genuine genomic context (i.e at the RIO1 locus) Gene-shuffling experiments showed that the (S > A)6 as well as the (S > D)6 mutant alleles complemented the deletion of the RIO1 wild-type copy However, growth rates of yeast cells harbouring the (S > A)6 mutant allele were significantly reduced FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4659 Rio1 protein kinase is regulated by CK2 M Angermayr et al Fig Functionality of the CK2 phosphorylation sites in yeast (A) Myc3-tagged Rio1 wild-type or mutant proteins were immunoprecipitated from yeast extracts with anti-(myc agarose) and incubated in the presence of [32P]ATP[cP], separated by SDS ⁄ PAGE and autoradiographed (B) Coomassie Brilliant Blue-stained gel (C) Quantitative evaluation of phosphate incorporation into the respective Rio1 proteins Values represent the average of three independent experiments, SD bars are given in the figure Fig CK2 phosphorylation stimulates Rio1p kinase activity (A) In vitro kinase assays were performed using recombinant affinity-purified His6-tagged wild-type or mutant Rio1p proteins (S > A)6 or (S > D)6, with histone H2B as a substrate (autoradiograph) (B) Coomassie Brilliant Blue stain of Rio1p input (C) Quantitative evaluation of phosphate incorporation into H2B (normalized to H2B input and to input of Rio1 proteins) Values represent the average of three independent experiments (three different purifications from E coli), SD bars are given in the figure 4660 (Fig 9A), indicating that the respective serine residues must be phosphorylated in vivo to give a biologically fully active Rio1p kinase The yeast strain carrying the mutant (S > D)6 allele behaved indistinguishably from wild-type, suggesting that the Rio1p kinase phosphorylated by CK2 is the fully functional form of the enzyme in vivo These findings obtained with the respective RIO1 mutant alleles corroborate the results obtained in vitro, i.e that the Rio1p kinase is moderately activated by CK2 phosphorylation also in vivo in the wild-type and that this activation accelerates cell proliferation These observations imply that lack of phosphorylation is disadvantageous for cell proliferation One possible reason for the slow growth of the nonphosphorylatable (S > A)6 mutant could be that these cells are impeded in entering or exiting from a certain FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS M Angermayr et al Fig Growth rates of RIO1 wild-type and mutant yeast strains and cell-cycle phase distribution (A) A yeast strain carrying a single genomic copy of the RIO1 (S > A)6 mutant allele is hampered in growth rate; the respective (S > D)6 mutant yeast strain is indistinguishable from the wild-type Two independent clones were tested in all cases Maximum deviations are indicated by bars (B) Logarithmically growing cells were stained with DAPI, photographed and evaluated according to the stages of the cell-division cycle as indicated below the diagram A total of 546 wild-type cells or 239 mutant (S > A)6 mutant cells have been analysed phase of the cell-division cycle Therefore, we tested, using light microscopy and after DAPI staining, whether the slow growth yields a cell-cycle phenotype Cells of the logarithmically growing wild-type or the (S > A)6 mutant strain were photographed and cells assigned to the respective phase of the cell-division cycle (similarly as described previously) [12] In the (S > A)6 mutant, the number of cells in the S phase having a small bud was drastically diminished to almost one third relative to wild-type or the (S > D)6 mutant, i.e 8% in the (S > A)6 mutant versus 22% in the wild-type, and the number of G1 cells was increased accordingly – 39% in Rio1 protein kinase is regulated by CK2 the (S > A)6 mutant versus 29% in the wild-type By contrast, G2 plus M phase cells were not affected significantly – 53% in the (S > A)6 mutant versus 49% in the wild-type (Fig 9B) However, we found a slight imbalance with respect to the distribution of G2 ⁄ M cells: the number of metaphase cells with a single DNA mass at the bud neck and the number of anaphase cells were increased slightly in the mutant (metaphase cells: 30.2% in the mutant versus 24% in the wild-type; anaphase cells: 4.5% in the mutant versus 3.2% in the wild-type), whereas the number of telophase cells was decreased slightly (18.3% in the mutant versus 21.8% in the wild-type) These slight imbalances are considered insignificant, in contrast to the differences observed with the distribution of G1 and S phase cells These observations suggest that (S > A)6 mutant cells, that fail to become phosphorylated, mainly have difficulties entering the S phase The reason for the accumulation of cells in the G1 phase may be due to the slightly lower kinase activity of the (S > A)6 mutant Rio1p kinase during the G1 phase relative to wild-type or the (S > D)6 mutant, thereby retarding entry into the S phase However, we obtained direct evidence that a different mechanism may play a role A first hint to this mechanism came from quantitative analysis of Rio1 protein concentrations in the two mutants In logarithmically growing cells of the (S > A)6 mutant the concentration of the respective Rio1 protein generally exceeded (approximately fivefold) that of the wild-type or the (S > D)6 mutant proteins (see Fig 10; log ¼ cycling cells) One possible explanation for this finding, which was further Fig 10 Phosphorylation of Rio1p by CK2 renders the protein susceptible to proteolysis.RIO1 wild-type, (S > A)6, or (S > D)6 mutant cells were arrested with a-factor, hydroxyurea (HU), or nocodazole (Noc) or left untreated (log) as a control in the presence of galactose as a carbon source (for details please refer to Experimental procedures) Cellular extracts were separated by SDS ⁄ PAGE, and the respective proteins were detected by western blotting (A) Immunoprecipitated myc3-tagged versions of Rio1p were detected by myc antibodies after SDS ⁄ PAGE in a western blot (B) Loading control The stable protein Aky2p served as an input control and was analogously detected by Aky2p antibodies derived from hen egg yolk FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4661 Rio1 protein kinase is regulated by CK2 M Angermayr et al pursued, is that the unphosphorylated version is stable, whereas the (S > D)6 mutant protein, mimicking the phosphorylated form (and obviously also wild-type Rio1p) is proteolytically degraded Generally increased proteolytic instability of Rio1p wild-type and the (S > D)6 mutant proteins relative to the (S > A)6 mutant was, however, not observed in unsynchronized cells (not shown) Therefore, we tested whether Rio1p and Rio1(S > D)6 mutant proteins are degraded at a certain stage of the cell cycle Such temporary instability may explain the lower steady-state concentration of the Rio1 mutant protein in the (S > D)6p version and wild-type Rio1p relative to (S > A)6p in asynchronous cells To test whether phosphorylated Rio1p is, in fact, degraded at a certain stage of the cell-division cycle, whereas the unphosphorylated form is not, we performed cell-cycle arrest experiments (see Experimental procedures) Logarithmically growing cells were induced by galactose (RIO1 alleles under the control of the GAL10 promoter) and simultaneously arrested by treatment with either a-factor (arrest before the G1 ⁄ S transition), hydroxyurea (S phase), or nocodazole (before onset of anaphase), respectively Cellular concentrations of Rio1p were measured relative to adenylate kinase (Aky2p), which is a constitutively expressed, stable protein [18] as a loading control (see Experimental procedures) Our results indicate that in the RIO1 wild-type and the (S > D)6 mutant the level of Rio1 protein is low to undetectable in the S phase but normal in G1 and during mitosis compared with cycling cells (Fig 10; traces of material detected after arrest with hydroxyurea may be attributable to nonarrested cells; 10–15%) However, the level of Rio1p is surprisingly high and constant in the (S > A)6 mutant and, most notably, not at all affected by the stage of the cell-division cycle (Fig 10), thus displaying significant resistance to proteolytic degradation These findings demonstrate that Rio1p and the (S > D)6 mutant proteins, mimicking the phosphorylated version, are degraded at the G1 ⁄ S transition, whereas the nonphosphorylatable (S > A)6 version is not Discussion The Rio1p kinase from yeast is essential and highly conserved from archaea to man and, thus, likely to serve an evolutionarily ancient, highly important, as yet unknown function [12–14,17,19] Therefore, it is of general interest to identify interaction partners or substrates and, as a consequence, the pathway(s) this kinase is involved in We have found that the catalytic a subunits of protein kinase CK2, Cka2p and to a lesser extent Cka1p, specifically interact with Rio1p We 4662 have shown that the C-terminus of Rio1p is essential and sufficient for this interaction We have also shown that Rio1p is a substrate for CK2 holoenzyme in vitro and, most likely, also in vivo Several combinations of serine mutations in the C-terminal portion of Rio1p proved the presence of six clustered CK2 phosphorylation sites with different affinities Neither the (S > A)6 mutant nor the 1–402 C-terminally truncated protein served as a substrate for CK2 (excluding the presence of additional CK2 sites N-terminal of position 402), whereas the C-terminal fragment Rio1-335–484p alone displayed strong wildtype-like phosphorylation by CK2 The C-terminal portion of yeast Rio1p displays a striking two-partite primary structure The part C-terminally adjacent to the catalytic domain is rich in serines and acidic residues (referred to as CK2 domain, positions 402–435), whereas the extreme C-terminus (positions 436–484) lacks serines and is highly positively charged (mainly lysines, referred to as K-domain, Fig 1A) It is noteworthy that the C-terminal domain of Rio1p is least conserved in evolution Archaea, that not have CK2, lack the CK2- and K-domains completely, but also among higher eukaryotic Rio1p orthologues high sequence divergence is observed in the C-terminal part Higher eukaryotes harbour two orthologues of Rio1p named the SUDD-type and the ad 034-type according to their first identification [13] The SUDD proteins have only a short stretch of basic amino acid sequences lacking C-terminal CK2 sites ad 034 proteins are more closely related to yeast Rio1p and have both a CK2- and a K-domain, although the direct sequence similarity is low We have cloned both types of human cDNAs, ad 034 and SUDD, as myc3tagged version and expressed them in yeast and E coli Neither, alone or together, complements RIO1 deficiency in yeast Nevertheless, both recombinant orthologous proteins are heavily phosphorylated by CK2 in vitro (M Angermayr, unpublished results) In vitro kinase assays with recombinant Rio1 proteins from yeast have revealed that the (S > D)6 mutation, mimicking permanent phosphorylation by CK2, stimulates Rio1p kinase activity about twofold with H2B as a heterologous substrate The same is true when the extent of Rio1p autophosphorylation of the respective mutant proteins isolated from yeast is compared, suggesting that phosphorylation of Rio1p by CK2 has only a minor, presumably modulating effect on the kinase activity of Rio1p Also with other substrates of CK2 that have been described in detail in the literature, very small physiological effects of phosphorylation by CK2 have been FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS M Angermayr et al reported Many of the targets of CK2 have been found to serve essential functions For example, CK2 increases the efficiency of transcription of the tRNA and 5S rRNA genes by RNA polymerase III due to phosphorylation of TBP within TFIIIB [20] Eukaryotic topoisomerase II is another target of protein kinase CK2 which is essential for viability However, the importance of CK2 phosporylation of topoisomerase II is not well understood, because mutation of the respective CK2 phosphorylation sites does not cause an obvious phenotype in yeast [21,22] Cyclin-dependent protein kinase from S cerevisiae, Cdc28p, is phosphorylated at a single serine residue by protein kinase CK2 [23] Lack of phosphorylation at this site affects neither Cdc28p kinase activity in vitro nor yeast growth rate, but leads to a slightly decreased cell size during the G1 phase [9,23]; by contrast, S > E mutation of this residue stimulates Cdc28p twofold, at least in vitro [9] Sic1p, the cyclin ⁄ CDC28 cell-cycle kinase inhibitor that prevents premature entrance into the S phase, is another interesting substrate of CK2, but phosphorylation by CK2 has little influence on its physiological function [10,24,25] The essential translational initiation factors, eIF2a (encoded by SUI2) [26] and eIF5 (encoded by TIF5) [27], are additional targets of CK2, but phosphorylation by CK2 of either eIF2a or eIF5 by CK2 is not essential for their respective functions A seeming exception of a low effect of CK2 site mutation is constituted by Cdc37p, a kinaseassociated molecular chaperone required in concert with Hsp90p in the regulation of the activity of several signalling protein kinases Mutation of the single CK2 site on Cdc37p is not lethal but severely impedes growth, presumably because of the additive negative effects on several important protein kinases [28,29] Taken together, there are many examples of proteins which serve important functions that are substrates of CK2, but mutational alteration of the respective CK2 phosphorylation sites has little effect or, at least, is not deleterious to cell viability This is more surprising as deletion of CK2 (in yeast the double deletion of CKA1 and CKA2) leads to inviability [6] However, the pleiotropy of CK2 may explain its indispensability Failure in CK2 deletion mutants of modulating a plethora of processes, although within narrow limits, is likely to be detrimental to cell life Thus, moderate activation of the Rio1p kinase upon phosphorylation by CK2 is in the same range as observed with most other substrates We have shown that mutant RIO1 alleles, in which all six CK2 phosphorylation sites have been mutated to alanine or aspartate, respectively, sustain yeast viability The (S > D)6 mutant behaves indistinguishably from wild-type indicating that Rio1p is heavily phosphory- Rio1 protein kinase is regulated by CK2 lated by CK2 in vivo In contrast, yeast cells harbouring exclusively the (S > A)6 mutant allele, a substrate that does not become phosphorylated by CK2, are severely hampered in growth rate In addition, we have observed a cell-cycle phenotype with this mutant Cytological approaches investigating cell-cycle stages reveal that this mutant yeast strain accumulates G1 cells, whereas the number of S phase cells is drastically diminished It may be noteworthy that we detected a slight imbalance of metaphase, anaphase and telophase cells as well Most significantly, this is in accordance with our previous observation that either depletion of Rio1p in the cell or the use of a weak D244E mutant allele leads to increased loss of minichromosomes and to the accumulation of both, large-budded M cells with a single DNA mass at the bud neck and large G1 cells, indicating that Rio1p is required for exit from mitosis and during G1 phase, but obviously not during S phase [12] However, in contrast to our previous Rio1p depletion experiments or the use of the weak active site mutant of Rio1 (D244E) in which inhibition of the entrance of anaphase was the most significant effect, we describe here with the nonphosphorylatable (S > A)6 mutant that the exit from the G1 phase is more pronouncedly retarded than the arrest in mitosis These findings indicate that Rio1p phosphorylation by CK2 mainly plays a role in the G1 phase conceivably by slightly increasing Rio1p kinase activity, but is less (or not) important during mitosis What might be the physiological basis of the moderate G1 arrest phenotype? By comparing the cellular concentrations of Rio1p and Rio1 mutant proteins, we were able to exclude that Rio1p or Rio1 (S > D)6p are proteolytically less stable per se than the (S > A)6 mutant protein This means that phosphorylation by CK2 causes no general signal for the degradation of Rio1p Rather, we observed that the cellular concentration of Rio1p and (S > D)6p is extremely low to undetectable in the S phase, indicating that phosphorylated Rio1p and the (S > D)6 protein are destined for degradation at the G1 to S boundary, whereas the (S > A)6 protein is not In this context, it may be relevant that the CK2 phosphorylation sites of Rio1p overlap a bona fide destruction box, an amino acid sequence rich in P, E, S and T residues which has been implied to be involved in the degradation of the respective protein in a ubiquitinand proteasome-dependent manner [30] In the case of Rio1pk, this potential degradation signal may be activated upon phosphorylation to act as a phosphodegron [31] and to effect cell-cycle phase-dependent proteolysis of Rio1p before entrance into the S phase This presumably occurs at the same time when other important G1-specific proteins (e.g Sic1p, Cln1p, FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4663 Rio1 protein kinase is regulated by CK2 M Angermayr et al Cln2p) [32]; are polyphosphorylated and destined for degradation through the ubiquitin-condensing E3-complex, SCF and the proteasome in order to establish commitment for progression to the S phase This would imply that Rio1p phosphorylation by CK2 constitutes a signal for its specific degradation upon G1 ⁄ S transition and that the absence of Rio1p during the S phase is conducive to cell proliferation, conceivably by accelerating passage through the G1 ⁄ S boundary The absence of wild-type Rio1p and, more obviously, of the (S > D)6 mutant protein during the S phase may be taken as evidence in favour of this interpretation In contrast, the concentration of the (S > A)6 mutant protein mimicking the unphosphorylated version is higher than wild-type (see Fig 10) The fact that growth is simultaneously impeded in the (S > A)6 mutant (see Fig 9) suggests that ridding of Rio1p before start of a new S phase might constitute a biologically important event and that the phosphorylated form of Rio1p is the more active and physiologically relevant In summary, we have described Rio1p as a substrate of protein kinase CK2 and provide evidence that phosphorylation of Rio1p by CK2 generates a signal for its degradation in a cell-cycle phase-specific manner Although proteolytic degradation of Rio1p is not a stringent prerequisite, the absence of Rio1p during the S phase promotes cell proliferation presumably by accelerating G1 to S transition Altogether, we believe that phosphorylation of Rio1p by CK2 has at least two biologically relevant consequences: (a) it increases Rio1 kinase activity and, conceivably, also controls interaction with other proteins (presumably during the G1 phase); and (b) it provides a signal for commitment for degradation of Rio1p before entrance of the S phase We have shown for the first time that the cellular concentration and activity of constitutively expressed Rio1p [15,16] are likely regulated through phosphorylation by CK2 Experimental procedures Plasmids and strains E coli expression vectors pQE32 (Qiagen, Hilden, Germany) or pGEX-4T-2 (Amersham Biosciences, Freiburg, Germany) were used to produce His6- or GST-tagged versions of Rio1p E coli strain BL21-Codon Plus-RIL (Stratagene, Heidelberg, Germany), which had been additionally transformed with the chaperonin-harbouring plasmid pREP4-groESL [33], served for recombinant expression of Rio1 proteins YEp351 or YEp352 [34] were used for protein expression in yeast strains WCG-4a (obtained from D H Wolf, University of Stuttgart, Germany) or BY4741 (Mat a, his3D1, leu2D0, met15D0, 4664 ura3D0) [35] (obtained from EUROSCARF, Frankfurt, Germany) Deletion strains (isogenic to BY4741) Y01428 (Dcka1) (Mat a, his3D1, leu2D0, met15D0, ura3D0, YIL035c:: kanMX4) and Y01837 (Dcka2) (Mat a, his3D1, leu2D0, met15D0, ura3D0, YOR061w::kanMX4) were obtained from EUROSCARF Integration plasmid pRS306 [36] was used to integrate myc3-tagged RIO1 alleles under the control of the GAL10 promoter into the genome at the URA3 locus Yeast strains carrying exclusively the RIO1 (S > A)6 or (S > D)6 mutant alleles in the genuine genomic context (i.e at the RIO1 locus under the control of the RIO1 promoter) were generated using yeast strain YMA69 (Mat a, rio1::HIS3, ade2–1, his3–11, 15, leu2–3, 112, trp1–1, ura3–1, can1–100 [pMA 221]) The haploid rio1-deletion strain was rescued by plasmid pMA221 which carried a RIO1 wild-type copy in the vector pGBDU-C1 [37] The coding regions of the (S > A)6 or (S > D)6 mutant alleles were amplified by PCR YMA69 was cotransformed with the respective PCR fragments and pFL36 [38] to have a selectable marker Transformants were replica-plated onto 5-flouroorotate-containing plates [39] to select against pMA221 Candidates were analysed by PCR and DNA sequencing In vitro mutagenesis and recombinant DNA methods Truncated versions of RIO1 were produced by PCR (Fig 1) RIO1 constructs were N-terminally equipped with a myc3-tag and expressed under the control of the GAL10 promoter from YEp351 in yeast strain WCG-4a CKA2 or CKA1, respectively, were amplified from genomic DNA by PCR, N-terminally fused to an HA3-tag, and expressed under the control of the GAL10 promoter from YEp352 Presumptive CK2 phosphorylation sites were mutated from serine to alanine or aspartate, respectively (QuikChange Site-Directed Mutagenesis Kit, Stratagene) (Fig 1) Co-purification experiments Yeast cells were disrupted by vortexing with glass beads in lysis buffer (50 mm Hepes-KOH, pH 7.25, 15% glycerol, 10 mm MgCl2, 0.1% NP-40, mm NaF, mm Na3VO4, mm phenylmethylsulfonyl fluoride, lgỈmL)1 each aprotinin, leupeptin and pepstatin) Myc3- or HA3-tagged proteins were immunoprecipitated with anti-(myc agarose) or HA antibodies (both from Santa Cruz Biotechnology, Santa Cruz, CA), respectively, or protein A–Sepharose (Sigma, Deisenhofen, Germany) in lysis buffer adjusted to 150 mm NaCl (final concentration) Immunoprecipitates were washed three times with 50 mm Hepes-KOH, pH 7.25, 15% glycerol, 150 mm NaCl, 10 mm MgCl2, 0.1% NP-40, mm NaF, mm Na3VO4, mm phenylmethylsulfonyl fluoride, lgỈmL)1 each aprotinin, leupeptin and pepstatin, and twice with 50 mm Hepes-KOH, pH 7.25, mm MgCl2, FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS M Angermayr et al 1.5 mm MnCl2 Samples were subjected to SDS ⁄ PAGE and analysed by MS or western blotting using antibodies directed against the myc- or HA-tag, respectively (Santa Cruz Biotechnology) Protein identification The Coomassie Brilliant Blue-stained protein spots of coprecipitates were excised from the gels and placed in a Multiscreen 96-well filter plate (Millipore, Bedford, MA) positioned on top of a vacuum manifold integrated into a Multiprobe II Ex robotic liquid-handling system (Perkin– Elmer, Wellesley, MA) The gel pieces were destained with alternating washing steps using 50 mm NH4HCO3 buffer and acetonitrile A vacuum was applied to drain the wells after each washing step Subsequently, trypsin (Roche Diagnostics, Mannheim, Germany) dissolved in 50 mm NH4HCO3 was added (enzyme to substrate ratio approximately : 10), and the plate was placed into an incubator at 36 °C overnight To elute the peptides, a 96-well receiver plate was positioned at the bottom of the Multiscreen filter plate in the vacuum manifold Gel pieces were incubated with acetonitrile for min, and the supernatant was eluted into the receiver plate under vacuum This elution step was repeated with 10% formic acid and acetonitrile The combined supernatants were spotted onto a MALDI target An aliquot (0.5 lL) of the sample was mixed on the target with 0.5 lL of the matrix solution (5 mgỈmL)1 of a-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile, 0.1% trifluoroacetic acid) and dried at room temperature Mass analysis was performed using a positive reflector mode with a deflection cut off range of m ⁄ z 800 on a 4700 Proteomics Analyser (Applied Biosystems, Framingham, MA) equipped with an Nd-YAG laser that produces pulsed power at 355 nm at pulse rates of 200 Hz One thousand laser shots were accumulated to produce one single spectrum Subsequently, high-energy MALDI-TOF ⁄ TOF CID spectra were recorded on selected ions from the same sample spot The collision energy was kV Air was used as collision gas The peptide mass fingerprints were submitted to a search at the NCBI protein database using mascot (http://www matrixscience.com) For unambiguous identification of the proteins, tandem MS analysis was performed on one or two peptides of the peptide mass fingerprints followed by a search of the NCBI protein database using mascot Purification of recombinant Rio1p from E coli His6-tagged Rio1p was expressed in E coli BL21 and purified as described [12,13] Expression of GST–Rio1p in E coli was induced by mm isopropyl thio-b-d-galactoside at 30 °C for 3.5 h Cells were harvested, incubated on ice in 50 mm Tris-Cl, pH 8.0, 15% glycerol, 15 mm KCl, mm MgCl2, mm NaF, mm Na3VO4, mm phenylmethylsulfonyl fluoride, lgỈmL)1 each aprotinin, leupeptin and Rio1 protein kinase is regulated by CK2 pepstatin, and mgỈmL)1 lysozyme for 30 and lysed by sonication The 15 000 g supernatant was incubated with glutathione agarose (Sigma) Sedimented complexes were washed three times with 50 mm Tris-Cl, pH 7.5, 100 mm KCl, 0.1% NP-40, mm MgCl2, mm NaF, mm Na3VO4, mm phenylmethylsulfonyl fluoride, lgỈmL)1 each aprotinin, leupeptin and pepstatin, twice in kinase buffer, and used for in vitro kinase assays In vitro kinase assays Recombinant human protein kinase CK2 holoenzyme (0.5 U, corresponding to ng; New England Biolabs, Frankfurt am Main, Germany) was incubated with purified recombinant enzymatically inactive GST–Rio1p as substrate in 20 mm Tris-Cl, pH 7.5, 50 mm KCl, 10 mm MgCl2 in the presence of lCi [32P]ATP[cP] (10 CiỈmmol)1; final ATP concentration 16.7 lm) at 30 °C for 30 Myc3-tagged Rio1 proteins were purified from yeast wild-type, Dcka1-, or Dcka2-genomic backgrounds and incubated in 50 mm Hepes-KOH, pH 7.25, 10 mm MgCl2 in the presence of lCi [32P]ATP[cP] (10 CiỈmmol)1; final ATP concentration 16.7 lm) at 30 °C for 30 Reactions were terminated by addition of gel-loading buffer and run on SDS ⁄ PAGE Gels were stained and dried for autoradiography Recombinant wild-type or mutant Rio1 proteins were incubated with 15 lg histone H2B (Roche) as a substrate in 50 mm Hepes-KOH, mm MgCl2, mm MnCl2 in the presence of lCi [32P]ATP[cP] (10 CiỈmmol)1; final ATP concentration 16.7 lm) at 30 °C for 30 Cell-cycle arrest experiments to test Rio1p stability Yeast strains carrying the respective myc3-tagged RIO1alleles under the control of the GAL10 promoter in the genomic context (the respective alleles were integrated at the URA3 locus with the help of the integration plasmid pRS306) were cultured on 2% glucose-rich medium, shifted to medium containing 2% raffinose as a carbon source for two generations and then 2% galactose (final concentration) was added At this point the respective yeast cultures were divided into aliquots and treated with a-factor (3 lgỈmL)1; with further addition of 1.5 lgỈmL)1 after 1.5 and 2.25 h to ensure a stable arrest), 150 mm hydroxyurea or 15 lgỈmL)1 nocodazole, respectively One untreated aliquot served as the control (cycling cells) Cells were pelleted, washed once in H2O and frozen in liquid nitrogen Subsequently, yeast cells were disrupted by vortexing with glass beads in the same buffer as described for the co-immunopurification experiments, protein contents were determined, and the samples subjected to SDS ⁄ PAGE and analysed by western blotting using myc antibodies (Santa Cruz Biotechnology) As a loading control, we used antibodies to detect Aky2p FEBS Journal 274 (2007) 4654–4667 ª 2007 The Authors Journal compilation ª 2007 FEBS 4665 Rio1 protein kinase is regulated by CK2 M Angermayr et al [18], a protein which is stable throughout the cell cycle and highly resistant to proteolytic degradation [40] Miscellaneous procedures Yeast were grown on standard media [41] and transformed as described by Gietz et al [42] Microscopy and cell cycle analyses were performed as described [12] Protein contents were determined by the method published by Bradford [43] Other molecular methods were performed according to standard procedures [44] or as recommended by the manu6 facturers References Glover CV III (1998) On the physiological role of casein kinase II in Saccharomyces cerevisiae Prog Nucleic Acid Res Mol Biol 59, 95–133 Ahmed K, Gerber DA & Cochet C (2002) Joining the cell survival squad: an emerging role for protein kinase CK2 Trends Cell 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