Just-in-time assembly of cell-cycle protein complexes by steepslope9876

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									                   Just-in-time assembly of cell-cycle protein complexes
                       Lars Juhl Jensen *, Ulrik de Lichtenberg *, Thomas Skøt Jensen *, Søren Brunak and Peer Bork
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                       European Molecular Biology Laboratory, Heidelberg, Germany                                                            Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark                                                                                                               * These authors contributed equally




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Nature Precedings : doi:10.1038/npre.2008.2273.1 : Posted 9 Sep 2008
Abstract                                                                                                                                           Identification of periodically expressed genes                                                                                                                                         Regulation differs between organisms
DNA microarray studies have shown that hundreds of genes                                                                                                                                                                                      Spellman et al. (1998)              0.5



are transcribed periodically during the mitotic cell cycle. We                                                      0.4                                                                                                                       Johansson et al. (2003)             0.4
                                                                                                                                                                                                                                                                                             S. pombe                                                            S. cerevisiae                 S. pombe
                                                                                                                                                                                                                                              Lu et al. (2004)
show here that although the protein complexes involved in this




                                                                                    Fraction of benchmark set identified
                                                                                                                                                                                                                                                                                  0.3
                                                                                                                                                                                                                                              de Lichtenberg et al. (2005)

process are largely the same among all eukaryotes, their                                                                                                                                                                                      Willbrand et al. (2005)             0.2



regulation has evolved considerably. Our comparative analysis                                                       0.3
                                                                                                                                                                                                                                              Qiu et al. (2005, 2006)
                                                                                                                                                                                                                                              Glynn et al. (2006)
                                                                                                                                                                                                                                                                                  0.1                                                                                     82                       103
of several large-scale datasets reveals that the identity of the                                                                                                                                                                              Ahnert et al. (2006)                  0                                                          H. sapiens                                                    A. thaliana
                                                                                                                                                                                                                                                                                                                                                                                   23
                                                                                                                                                                                                                                                                                  0.4

                                                                                                                                                                                                                                              Lu et al. (2006)                               H. sapiens
periodically expressed proteins differs significantly between                                                                                                                                                                                 Liew et al. (2007)
                                                                                                                                                                                                                                                                                                                                                                         14                            5
                                                                                                                                                                                                                                                                                  0.3


organisms, yet the regulated subunits of each protein complex                                                       0.2
                                                                                                                                                                                                                                              Cho et al. (1998)
                                                                                                                                                                                                                                              Zhao et al. (2001)                  0.2

are expressed just prior to its time of action. Moreover, we                                                                                                                                                                                  Langmead et al. (2003)
                                                                                                                                                                                                                                                                                                                                                              86               3               2             26
demonstrate that these changes in transcriptional regulation
                                                                                                                                                                                                                                                                                  0.1
                                                                                                                                                                                                                                              Wichert et al. (2004)
                                                                                                                                                                                                                                              Luan & Li (2004)
have co-evolved with post-translational control; loss or gain of                                                                                                                                                                              Ahdesmaki et al. (2005)
                                                                                                                                                                                                                                                                                     0

                                                                                                                                                                                                                                                                                                                                                                                    5
cell cycle-regulated transcription of specific genes is often
                                                                                                                       0.1
                                                                                                                                                                                                                                              Chen (2005)
                                                                                                                                                                                                                                                                                  0.3
                                                                                                                                                                                                                                                                                             A. thaliana
                                                                                                                                                                                                                                                                                                                                                                          10                       6
                                                                                                                                                                                                                                              Andersson et al. (2006)
mirrored by changes in phosphorylation of the proteins they                                                                                                                                                                                   Xu et al. (2006)
                                                                                                                                                                                                                                                                                  0.2
                                                                                                                                                                                                                                                                                                                                                                               1               7
encode. Our results indicate that many different solutions have                                                                                                                                                                               Random

                                                                                                                                                                                                                                                                                                                                                                                    8
                                                                                                                                                                                                                                                                                  0.1

evolved for assembling the same molecular machines at the                                                                  0
                                                                                                                               0                       200            400           600                     800       1000       1200
right time during the cell cycle, involving both transcriptional                                                                                                 Number of genes identified                                                                                          0
                                                                                                                                                                                                                                                                                         0       400        800       1200



and posttranslational layers that jointly control the dynamics
of biological systems.




                                                                                                                                                                                                                                                                           3
                                                                                                                                                                                Co-evolution of transcriptional and posttranslational cell-cycle regulation
                                                                                                      Linking transcription and phosphorylation
                                                                                                                                                                                                                                                                                                                                                Dynamic proteins
Transcriptional regulation is rarely conserved
                                                                                                                                                                        Dynamic proteins
                                                                                                                                                                                                                                                                                                                                               with static orthologs




                                                                                                                                                                                                                                                                                                                                                                                                               Proving co-evolution of transcriptional
                                                                                                                                                                                                                                        Organism             Data source                                          P-value

To obtain comparable sets of transcriptionally regulated genes from                                                                                                                                                                     H. sapiens           LTP CDK                                              7 • 10-9




                                                                                                                                                                                                                                                                                                                                                                                                                  regulation and phosphorylation
                                                                                                                                                                                                                                                             HTP S/T                                              4 • 10-16
distantly related eukaryotes, we reanalyzed the existing cell cycle                                                                                                         P
                                                                                                                                                                                                             P                                                                                                                                   P
                                                                                                                                                                                                                                        S. cerevisiae        HTP CDK                                              2 • 10-55                                      P
gene expression data and found 600 periodically expressed genes                                                                                                                             P
                                                                                                                                                                                                                                        S. pombe             NetPhosK                                             4 • 10-11                               P
in H. sapiens4, 600 in S. cerevisiae1,2, 500 in S. pombe3, and 400 in                                                                                                                                                                   A. thaliana          NetPhosK                                             0.03
A. thaliana4. Our benchmarks show that these gene lists are more                                                                                                                     P
                                                                                                                                                                                                             P
conservative than those originally proposed, yet achieve better                                                                                                                                                                                                                                                                                                  P
sensitivity (estimated to be 80–90% for human and the two yeasts,                                                                                                                                                                       Organism 1           Data source         Organism 2                       P-value
but only 50% in the plant). Surprisingly, we also discovered that                                                                                                                                                                       H. sapiens           LTP CDK             S. cerevisiae                    4 • 10-3
                                                                                                                                                                                                                                                                                                                                                     Organism 1                      Organism 2
most of the published methods for identifying periodically expressed                                                                                                                                                                                                             S. pombe                         0.02
perform worse than the original analysis by Spellman et al.1 (Fig. 1)                                                                                                                                                                                                            A. thaliana                      0.06
                                                                                                                                                                                P                                                                            HTP S/T             S. cerevisiae                    0.07
We further assigned genes with a common descent to orthologous                                                                                                                                                                                                                   S. pombe                         7 • 10-4
groups, of which 381 groups contain orthologs from all four                                                                                                                                                                                                                      A. thaliana                      6 • 10-4
                                                                                                                                                                                                                                        S. cerevisiae        HTP CDK             H. sapiens                       2 • 10-3
organisms and have at least one periodic member. We found that                                                                                                                                                                                                                   S. pombe                         4 • 10-3
periodicity is poorly conserved across the four organisms, meaning                                                                                                                                                                                                               A. thaliana                      9 • 10-3
that although the protein sequences are conserved through                                                                                                                       P
                                                                                                                                                                                                                                        S. pombe             NetPhosK            H. sapiens                       0.08                                           P

evolution, their transcriptional regulation during the cell cycle is not                                                                                                                                                                                                         S. cerevisiae                    0.02
(Fig 2). The large differences observed cannot be explained by the                                                                                                                                                                                                               A. thaliana                      0.2                           Static proteins with
                                                                                                                                                                            Static proteins                                                                                                                                                     dynamic orthologs
quality of the gene expression data or the orthology analysis4.




Multiple layers of regulation have co-evolved                                               Peak time                                                                                                                                  DNA polymerases                                                                                                   Deoxynucleotide biosynthesis
The agreement between the timing of transcription and the timing of                                                        H. sapiens                                                                            H. sapiens                   S. cerevisiae                                  S. pombe                                            H. sapiens                    S. cerevisiae                 S. pombe




                                                                                                                                                                                                                                                                                                                              4
action of the protein products is not necessarily to be expected as                                                                               M/G1

phosphorylation is known to be at least as important for regulating


                                                                                                                                                                                                                                                                                                                              RNR complex
                                                                                                                                                                                                                                POLE                                POL2                                          cdc20                                       RRM1                 RNR1                                        cdc22
                                                                                                                                                                                    DNA polymerase ε




cell cycle complexes. In this regard, we have previously noted that                                                                                                                                                 POLE2                            DPB2                                       dpb2
                                                                                                                                                                                                                                                                                                                                                                                     RNR3
                                                                                                                                                                                                                                                                                                                                                 P
periodically expressed S. cerevisiae proteins tend to also be
                                                                                                                                         15–19 hr
                                                                                                                                                                                                                                                                                                                                                      RRM2                         RNR2                                        suc22
                                                                                                                                                                                                                        POLE3                     P          DPB3                                       dpb3
phosphorylated. To generalize this observation, we annotated the                                                                                                                                                                                                                                                                                         RRM2B                       RNR4
                                                                                                                           S
complexes with information on phosphorylation by CDKs, and found                                                                                                                                            POLE4                            DPB4                                    dpb4


that the dynamic subunits are indeed three times as likely to be                                                                                                                                                                                                                                                                                                                     YLR437C                                  spd1
                                                                                                              S. cerevisiae
targeted by phosphorylation as the static ones (P < 0.001)4.
                                                                                                                                                                                                                                                                                                                              Inhibitors




                                                                                                                                                   M/G1                                                                         POLA                     P          POL1                                          pol1
                                                                                                                                                                                    DNA polymerase α




To assess the evolutionary significance of this correlation, we                                                                                                                                                     POLA2                            POL12                                      spb70
                                                                                                                                                                                                                                                                                                                                                                                        SML1

applied two proteome-wide statistical tests to several independent
                                                                                                                                                  1–2 hr
sets of known and predicted phosphoproteins, which revealed a
                                                                                                                                                                                                                        PRIM1                                PRI1                                          spp1
                                                                                                                                                                                                                                                                                                                              Kinase




highly significant overrepresentation of dynamic proteins among the                                                                                                                                        PRIM2A                             PRI2                                       spp2                                                                                           DUN1

CDK substrates4 (Fig. 3). To check if this reflects that loss or gain of
                                                                                                                                                             S


transcriptional regulation of a gene is correlated with loss or gain of                                                    S. pombe
                                                                                                                                                                                                                                                                                                                              Nuclear import




phosphorylation of the corresponding proteins, we compared the                                                                                                                                                                  POLD1                               CDC2                                          cdc6                                                              WTM1
                                                                                                                                                                                    DNA polymerase δ




                                                                                                                                                   M/G1
dynamic proteins with static orthologs to static proteins with
                                                                                                                                                                                                                                                                                                                                                                                                                              kap111
                                                                                                                                                                                                                    POLD2                            HYS2                                       cdc1
                                                                                                                                                                                                                                                                                                                                                                                   KAP122
dynamic orthologs and found that transcriptional regulation and                                                                                                   S                                                     POLD3                     P         POL32                                       cdc27

phosphorylation have indeed co-evolved4. Similar, we find that                                                                                    2–3 hr
                                                                                                                                                                                                           POLD4                                                 P                   cdm1
dynamic proteins are often subject to ubiquitin-mediated targeted
degradation by the proteasome4,5.




The just-in-time assembly principle




                                                                                                                                                                                                                                                                                                                                                                               5
In order to find a model, which could explain these apparently
                                                                                                                                                                                                                                                                                                                                                Dynamic
conflicting observations, we turned to a principle for regulation of                                                                                                                                                                                                                                                                             protein
cell-cycle protein complexes in budding yeast that we uncovered                                                                                                                                                                         Inactive                           Inactive                                                                                                Periodically transcribed gene
recently. By constructing a temporal interaction network of the cell                                                                                                                                                                    complex                            complex
cycle, we found that protein complexes were generally formed as a
combination of static (constitutively expressed) and dynamic
(periodically expressed) proteins2. The latter showed a clear
tendency to be expressed right before the complex is known to
become active and thus, by their change in abundance, control the                                                                                                                                                            Active                                                            Active
assembly of the functional complex. This mechanism, which we                                                                                                                                                                complex                                                           complex
termed “just-in-time assembly”, is illustrated in Fig. 5. When                                                                          Protein
studying a set of protein complexes that are conserved across all                                                                     degradation
four organisms, we saw that the identity of the dynamic subunits
within each complex has changed during evolution (Fig. 4).4 The
just-in-time assembly model also implies that the dynamic proteins
must be eliminated at a later stage in the cell cycle, explaining the
observed co-evolution between different layers of regulation.



References
1
  U. de Lichtenberg*, L.J. Jensen*, A. Fausbøll, T.S. Jensen, P.                                                       Regulation of degradation                                                                                                                                                                                                                 Regulation of localization
Bork and S. Brunak, ”Comparison of computational methods for
the identification of cell cycle regulated genes”, Bioinformatics, 21,                                                                                                      Inactive                                                                                                                                                                                                                   Phosphorylation
                                                                                                                                                                            complex                                                                  Regulation of assembly
1164–1171, 2005
2
 U. de Lichtenberg*, L.J. Jensen*, S. Brunak and P. Bork,
                                                                                                                                                                                                                                                                                                                                                                                                                                 P
“Dynamic complex formation during the yeast cell cycle”, Science,
307, 724–727, 2005                                                                                                                                                                                                                                                                               Phosphorylation
                                                                                                                                                                                                                                                                                                                  P
3
  S. Marguerat*, T.S. Jensen*, U. de Lichtenberg, B.T. Wilhelm,
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L.J. Jensen and J. Bähler, “The more the merrier: comparative                                                                                                                                           Active
                                                                                                                                                                                                                                            complex                                                                                                   complex
analysis of microarray studies on cell cycle-regulated genes in                                                                                                                                        complex                                                                                                                                                                                          Subcellular
fission yeast”, Yeast, 23, 261–277, 2006                                                                                                                   P                                                                                                                     P                                                                                                                     translocation
4
 L.J. Jensen*, T.S. Jensen*, U. de Lichtenberg*, S. Brunak and                                                                                        Protein
                                                                                                                                                    degradation
P. Bork, “Co-evolution of transcriptional and posttranslational cell
cycle regulation”, Nature, 433, 594–597, 2006
                                                                                                                                                                                                                                                                              Active
5
 U. de Lichtenberg, T.S. Jensen, L.J. Jensen, S. Brunak and                                                                                                                                                                                                                  complex                                                                                            Active
P. Bork, “Same molecular machines, different regulation”, Cell                                                                                Phosphorylation                                          P                                                     P                                                                                                       P         complex             P
Cycle, advance online publication, 2007

								
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