Through the cell cycle: cyclin dependent kinases

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1. Introduction to the Cdks:The aim of our study

2.Analysis of the protein sequence

2.1 Identification of the family members.
2.2 Alignment of the homologue sequences and phylogenetic evolution of the members.
2.3 Characterization of the consensus patterns reported in the protein family.
2.4 Proteins interacting with Cdk2
2.5 Common Blocks in the human Cdk's family
3.Phylogenetic distribution of the protein subject of our study

4.Analysis of the genes encoding the protein we study

                    4.1 Genomic location of the Cdk2 gene
                    4.2 Exonic compositions of the Cdk2 gene
                    4.3 Looking for undiscovered members of the family ...
                    4.4 Transcription of the Cdk2 gene: its promoter
                    4.5  Study of the Cdk2 splicing variants
                    4.6 SNPs analysis

5.Short structure analysis:

                 5.1 Structure presentation
                    5.2 Protein domains and phosphorylation sites




The cyclin-dependent kinases (Cdks) are a family of protein kinases whose members are small proteins (~34-40 kD) containing little more than the catalytic core sequences shared by all protein kinases. They are members of the Ser/Thr protein kinase super family and the cdc2/cdkx subfamily. By definition, all Cdks share the feature that their enzymatic activation requires the binding of a cyclin regulatory subunit. In most cases, full activation also requires phosphorylation of a threonine near the kinase active site. (See further information about 3D structure below).


Although originally identified as enzymes that control cell cycle events, members of the Cdk family are involved in other cellular processes as well. Animal cells, for example, contain at least nine Cdks, only four of which (Cdk1, 2, 4, 6) are involved directly in cell cycle control. Another family member (Cdk7) contributes indirectly by acting as a Cdk-activating kinase (CAK) that phosphorylates other Cdks. Cdks are also components of the machinery that controls basal transcription by RNA polymerase II (Cdk7, 8, 9) and are involved in controlling the differentiation of post mitotic neural cells (Cdk5).Based on the fact that all the members of the Cdk family show a great homology (see BLAST results), our study will consider them all eventhough they have different functions.


In the yeasts S. pombe and S. cerevisiae, all cell cycle events are controlled by a single essential Cdk, which are named as cdc2 and csc28 respectively. Cell cycle events in multicellular eukaryotes are controlled by two Cdks, known as Cdk1 and Cdk2, which operate primarily in M phase and S phase, respectively. Animal cells also contain two Cdks (Cdk4 and Cdk6) that are not essential components of the core cell cycle control system but are important regulators of entry into and exit from the cell cycle in G1.


Cdk function has been remarkably well conserved during evolution. It is possible, for example, for yeast cells to divide normally when their cdc2 / cdc28 gene is replaced with the human Cdk1 gene. This and other evidence clearly illustrates that Cdk function, and thus the function of the cell cycle control system, has remained fundamentally unchanged over hundreds of millions of years of eukaryotic evolution.


The aim of our study is to understand the relationships among the different Cdk members, both among the human but also different species along the evolutionary trend. Taking as a starting point the human Cdk2, we will first study the human homologues, and later on, the homology existent in further well characterized species. the most part of the study will be perform upon the protein sequences, due to the higher functional constrictions that occur on them.  Furthermore, we will also study the location of the human homologues within the whole human genome, the splicing variants and a few topics about the 3d structure of the Cdk2 as a model of the whole family


2.1 Identification of the family members.

Click in this link to visit the NCBI entrance to the original protein sequence that we used in order to start our work or here to access to the FASTA file.

We initially faced the determination of the other members that belong to the same protein family by searching likelihood scores among the different sequences. To this porpoise, tools available in the existent databases were used. The first step of the analysis of our protein was a Blast search with our initial amino acid sequence.

The results of the blast search show the existence of multiple homologous sequences, most of them related to the control  of the cell cycle. A clear homology is found among seldom human sequences (click here to see results table). Furthermore, we report the existence of many analogous protein in other species (see Cdk's phylogenetic distribution below).

In these initial studies, we just considered the human proteins which clearly belong to the family. We set the appearance of false positive results at the blast search around the following E-values:

1.  The first human protein without Cdk function is KPT1_HUMAN Q00536 a SER/THR–PROTEIN KINASE  from the PCTAIRE family, which shows to have an E-value= 2e-86 with a score = 317 in our blast search.

2.  The last human protein which belongs to the Cdk family is CDK8_HUMAN E-value= 2e-47 score = 178.


3.  In addition, we also located the first non human protein without Cdk function, which is called CC2H_DICDI P34117 CDC2-LIKE SER/THR–PROTEIN KINASE CRP. Its E-value = e-111 and presents an score = 298. (further information about the phylogenetic distribution of the Cdk family protein in the second part of our work).

2.2 Alignment of the homologue sequences and phylogenetic evolution of the human members.

The most representative sequences selected from the protein blast search, allowed us to see that the family of human Cdk is formed by 10 homologous proteins which have different roles during the cell cycle.

The next step was, to perform the alignment of these ten sequences named above.  The multiple alignment of the human protein sequences was done with clustalW - note that we used the GONNET series matrices to generate the multiple sequence alignment. These matrices were derived using almost the same procedure as the PAM matrices (Dayhoff) but are much more up to date and are based on a far larger data set. They appear to be more sensitive than the Dayhoff series and therefore, we selected them as the most adequate for our porpoise.

The results of this alignment show the most conserved residues present in the proteins.

Based on the results of the clustalW alignment, we built a neighbour phylogeny tree with the 10 homologue sequences found. This tree was done with PHYLIP tools. We used the program Neighbour in order to build the tree, and then, further work was done to evaluate the strength of the branches in it. The results of the bootstrap analysis do not support the ones obtained with Neighbour. Nevertheless, we suggest bootstrap as the most trustworthy analysis (click here to see the resulting results and tree).

 2.3 Characterization of the consensus patterns reported in the protein family.
The consensus patterns for these family sequences were searched in the InterPro page. Surprisingly, there are no specific consensus for the members of the Cdk family. However, two consensus sequences were obtained:


The position of these consensus patterns in the tyrosine kinase domain of the human Cdk2 was studied by manual direct observations and we could check their presence in the aligned sequences of the hole human Cdk family.
Moreover, not only the Cdks have been conserved in Evolution, but also the protein target specificity of the Cdk catalytic subunits has been well conserved . Cdk1 and Cdk2 from all species prefer protein substrates in which the phosphorylated serine or threonine residue is followed by a proline; it is also highly favourable for the target residue to have a basic amino acid nearby.

Finally, we show here the consensus of the phosphorylation sites of Cdk1 and Cdk2, which are typically defined as S/T*-P-X-K/R, where 'S/T*' indicates the phosphorylated serine or threonine, X represents any amino acid, and 'K/R' represents the basic amino acids lysine or arginine.

2.4 Proteins interacting with Cdk2

To see which proteins were interacting with the Cdk2, we consulted the Database of interacting proteins. (Click here to consult the results and the experiments done in order to confirm such an interaction).

2.5 Common Blocks in the human Cdk's family

The results of this search reported us a list of common sequences in our 10 members Cdk family which can be used to identify them as an homologous group.




In order to find out the presence of our protein family among the biological kingdoms, we performed a blast search against the reference organisms whose genome have been already sequenced (or with organisms from which we could access to the draft sequence of their genome).  As we did before with the human sequences, the alignment was performed taking in account those showing the greater E-values.

In order to settle the phylogeny of the cdk related proteins that we had selected from the blast search, we first built a NJ tree with the sequences of the different taxa. As previously reported, the tree was done with Neigbor and further bootstrap strength analysis was perform. The results show a strong value of all the branches shown in the tree.

The tree clearly shows the existence of a higher homology in each Cdk type, beyond the taxonomic differences. Thus, the Cdks present in many of our organisms of study are more similar among them, than two given Cdks in a determinate organism. Therefore, we claim the existence of a high conserved orthologous homology among the Cdk family.




4.1 Genomic location of the Cdk2 gene

We first obtained the FASTA genomic sequence. (In this file, coding fragments of the sequence appear in capital letters while non coding fragments are shown in lowercase letters.)
NCBI allowed us to find out the location of the Cdk2 human gene in the 12th chromosome. Concretely in between the bands 12q13.11 and 12q13.12. SeeMaster map genes in cr.12.

4.2 Exonic compositions of the Cdk2 gene

We next determined the exons composition of the gene. With this objective we performed a search in the Human Genome Project Working Draft. The results of this search showed us the predictions of the introns and exons present in our sequence. Acembly, Ensembl, Fgenesg++ and Genscan, were used to develop these predictions. An alignment of the different programs predictions is attached. All of them display the same 7 exons distribution which is, in fact, supported by the already known genes reported in RefSeq from NCBI. Only the Ensembl prediction presents one more exon. Which can in this conditions be excluded.

Moreover we run the Geneid. This results (see results plot) were compared with the previously linked alignment in order to obtain further conclusions. As it can be seen, Geneid identifies only 6 from the 7 real exons from Cdk2_human gene.

A slightly comparison of the exon distribution among the different homologues present in human and yeast show that exon I is longer in CDK2 than in the characterized CDC2 genes and is conserved in X. laevis Cdk2. Other differences between the CDK2 and the CDC 2 gene structure include two additional introns located at amino acids 105 and 196 of the human CDK2 gene that are not present in the Sacchromyces pombe CDC2 gene.

4.3 Looking for undiscovered members of the family ...

We finally tried to answer the question: would it be possible that further members of this family have not been yet characterized?

Once located the Cdk2 gene in the human genome, we looked for homologous genes to the Cdk2 in the hole genome draft. The results showed us many significant alignments. If the identification number (example with the first sequence obtained AC025162.31.80081.104328) of this DNA sequences is clicked, you will be directly delivered to the respective alignments page where the score of the different fragments can be evaluated. Once there, by clicking again in the same ID number, you will be shown the complete information known about the homologous fragments obtained in the search as exactly location of the DNA region, it's gene content, the identified RNAs and the proteins translated.

In order to view quickly all these results, consult the table which sums up the most relevant information obtained in e! Ensembl Human:

4.4 Transcription of the Cdk2 gene: its promoter

According to the data previously reported in the literature, five transcription start sites - spread over a 72-bp upstream region-  were identified.

However, no consensus TATA box was identified in the entire upstream sequence. Thus, this promoter falls into the category of TATA-less promoters similar to all other cell cycle genes analyzed to date including: cdc2, cyclin A, cyclin D1, cyclin D2 and cyclin D3, as well as Xenopus laevis Cdk2.

A YY1 box, which in some TATA-less promoters is responsible for determining the transcription start site, is present just upstream from the three start sites located at positions +1, -5, and -9.

An Sp1 site was identified upstream of each of the remaining transcription start sites (-33 and -71), suggesting that these Sp1 regions may be responsible for localizing the start of transcription at these sites . In addition, other putative transcription factor binding sites were also identified, such as a c-Myb binding site, or two putative p53 binding sites. It is perplexing to assume that p53 would induce CDK2 since this induction would most likely result in an accelerated cell cycle rather than a cell cycle arrest.

4.5  Study of the Cdk2 splicing variants

In humans - like in mice, rats and hamsters -  two variants of the Cdk2 were identified as a result of alternative splicing. The two resultant proteins reported do conserve their kinase function with small efficience variations:
    • Variant 1 (NM_001798) (cdk2beta) which contains an extra fragment within the coding region
    • Variant 2 (NM_052827) (cdk2alpha) which encodes a protein that lacks 34 aa internal fragment 
The FASTA files Variant1 and Variant2 from the mRNAs where used to perform a Clustalw alignment in order to be able to see their common and the singular fragments. Results are attached.

As it can be observed, the fragment missing in the second splicing variant corresponds to the nucleotides 722 to 824 which codify for 34 aminoacid. This peptide sequence was translated using ORF Finder from NCBI to get the corresponding peptide chain:

   H  H  L  S  K  D  A  A  Q  A  P  D  V  H  S C  G  I  I  F  A  A  Q  E  D  F  R  S  S  V P  Q  G  H

Once known the difference in the aminoacid sequence, our intention was to locate these 34 aminoacid within the 3-D structure of the protein ( human Cdk2) in order to know in which domain was contained. Unfortunately, all the crystallography experiments available in PDB have been done with the Variant2, and therefore our work was frustrated.

However, we followed this goal with news techniques. Below, a representation of the genomic DNA and the isolated mRNAs overlapping regions are shown. This way, we located this splicing variant in the 5th exon.

Small nucleotide differences in exons 1,5,6 and 7 can be displayed at the Evidence Viewer site.

We tried to discover if these exon distribution patron was also followed by other genes in the family. With this intention we performed the same search for the CDK3 and CDK8, respectively the ones showing the better and worst E-values in the initial table. On one hand, and from what can be seen, we deduce that some genes conserve a relatively similarity to the genomic structure of CDK2. CDK3, as CDK2, presents a 7 exons distribution and the position of these exons reminds to the one in CKD2. On the the other hand CDK8 presents a much more diverse exon structure, with 14 exons that seem much less robust

4.6 SNPs analysis

Most of the results obtained correspondent with DNA zones where no gen belonging to the Cdk family was identified. Some homologous fragments to the Cdk2 sequence where found in fragments where no genes at all had been described. Two genes (Cdk1 and Cdk4), already known to belong to the family did not appear in the results when the search was performed with the standard options. Two other genes appeared showing great homology, CdkL2 and CdkL3. This made us realize there are 3 more members - Cdkl1, CdkL2 and CdkL3 - belonging to the Cdk's family which we hadn't  take in account. The hole of this work will be done without including these in the searches.

Looking at these results we conclude it could be possible that some genes - or pseudogenes - belonging to this family haven't been identified yet. Only the progression of the research on genes isolation and characterization will allow these theoretical genes (or high homology sequences) to be accepted or refused.

A SNPs (single strand polymorphysms) analysis was perform in order to characterize the nucleotide variability within the human species. 
(According to the NCBI SNP Database) not withdrawn, single nucleotide mutations are shown here:
Genomic Data Transcription Data
SNP ID Contig Accession Position in Contig Orientation 5' Flanking Sequence* Nucleotide Change 3' Flanking Sequence* Validation Type mRNA Accession Protein Accession ORF Position in Protein Amino Acid Change
rs2069413 NT_009458.6 1040319 reverse TCTTCCAGGATGTGA C/G CAAGCCAGTACCCCA + coding-nonsynon XM_049150 XP_049150 2 200 T/S
rs2069406 NT_009458.6 1042343 reverse CTGCATCTTTGCTGA G/A ATGGTATGGAGGCTT + coding-synon XM_049150 XP_049150 3 105 E/E
rs2069400 NT_009458.6 1044292 reverse TTCACCTTCACCAGG C/A GGCTTTACTTACCTA + mrna-utr XM_049150 -- -- -- --
rs2069401 NT_009458.6 1044233 reverse TAATATATCCAAAAA C/T CACACCCTGACTACC + mrna-utr XM_049150 -- -- -- --
rs2069397 NT_009458.6 1044952 reverse GGGTTCCCAGGCCCC C/A GCTCCAGGGCCGGGC + mrna-utr XM_049150 -- -- -- --
rs2069398 NT_009458.6 1044831 reverse CAAGTTGACGGGAGA G/A GTGGTGGCGCTTAAG + mrna-utr XM_049150 -- -- -- --
rs2069399 NT_009458.6 1044336 reverse GGGGCTACTCCTGCA T/C TTTTTCCCCTCCATT + mrna-utr XM_049150 -- -- -- --
rs2069402 NT_009458.6 1043355 reverse TAACAAACATTTTTT C/T AATGCACAGGATGTA + intron XM_049150 XP_049150 -- -- --
rs2069403 NT_009458.6 1042757 reverse ttgggaggctgaggt G/A ggaggatcacttgag + intron XM_049150 XP_049150 -- -- --
rs2069404 NT_009458.6 1042709 reverse atcatgggcaacata G/A cgagaccccatctct + intron XM_049150 XP_049150 -- -- --
* Lower case letters indicate repetitive or low-complexity sequence


The SNPs distribution, as expected shows a higher presence of single nucleotide polymorphisms in the non coding / non translated regions than in the regions coding for the protein. But  we should consider the contig composition.

SNPs in NT_009458.6 Length SNPs presence Normalized values
SNPs in introns and utr mRNA 73,45%            80%             1.0891
SNPs in coding regions 26,55%           20%             0.7533




5.1 Structure presentation

The Cdk2 protein is a serine/threonin kinase involved in the cell cycle regulation. This protein interacts with the cyclins A, D and E and it's activity is maximal in phases S and G2.
For further illustration of the Cdk2 (variant2) protein structure, the secondary and1B39 tertiary ( PDB) structure of the protein can be observed. (The magnesium ion present in the structure is due to the crystalization conditions of 2.5 mM ATPand 5 mM MgCl2).

 We compared, manually, the regions of less similarity from the Clustalw run with all the human Cdks and the the secondary structure from the protein. We could appreciate that, as expected, the regions showing a greater divergence level were located in the zones in between the alpha helix and the beta sheets, were variations are allowed without representing a lose of activity.

5.2 Protein domains and phosphorylation sites

This enzyme is formed by two domains. A N-terminal beta sheet domain where the phosphorylation activity is located and a Cterminal multi-helix domain with kinase activity.

Cdk2 requires association with cyclins and phosphorylation by  CAK at Thr-14 and Tyr-15 for being active. The inactivation of the enzyme is done by the phosphorylation of the Thr-160 site. All these sites can be observed in an amplified version of the previous picture.




Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity. Brown NR, Noble ME & Co. J Biol Chem 1999 Mar 26;274(13):8746-56


Characterization of the Human Cyclin-dependent Kinase 2 Gene. Promoter analysis and gene structure. Dov Shiffman, Eric E. Brooks, Alan R. Brooks, Christine S. Chan, and Peter G. Milner
The Journal of Biological Chemistry Vol. 271, No. 21, Issue of May 24, pp. 12199–12204, 1996


The Differential Catalytic Activity of Alternatively Spliced Cdk2a and Cdk2b in the G1/S Transition and Early S Phase. Taeg Kyu Kwon, Meredith A. Buchholz, Do Youn Jun, Young Ho Kim, and Albert A. Nordin
Experimental cell research 238, 128–135 (1998)

Programs and information available in this pages were used no perform most of the searches.

To perform the alignments the next pages were used:

The interactions between Cdk2 and other proteins were obtained from:

The consensus patterns were defined with the help of:

The phylogenetic trees were performed using many programs at:

Exons prediction and alignment was searched at:

To analyse the secondary and tertiary structure we used: