FIND ARTICLE

Volume: 
Supplement: 
19
Date of issue: 

In response to various external and internal stimuli stability , and thus the amount , subject to a number of regulatory proteins in eukaryotic cells of "dynamic " control precision by proteolysis. In general, proteins targeted for degradation in a specific way wyznakowywane by complexation with ubiquitin , a small (76 AA) commonly occurring globular protein . In cascade occurs with the participation of ATP protein ubiquitination involving at least three enzymes : ubiquitin activating enzyme (E1) , an enzyme combines with the activated ubiquitin (E2 ) and conveying it to a protein substrate , either directly , or more frequently , in cooperation with a ubiquitin ligase (E3 ) . Ubiquitin ligases are complexes of some ( few ? ) Proteins involved in the precision of recognition and binding of protein substrates and their translocation into the 26S proteasome , which are degraded to short peptides. In recent years, " discovered " are getting a new protein , which is attributed to the functions of the ubiquitin ligases , and thanks to the knowledge of the structure and operation of individual subunits becomes possible to eject conclusions as to their homology in certain ligazach .
The degradation of most of the proteins involved in the control of the course of the successive phases of the cell cycle occurs primarily in the 26S proteasome. In particular, the cyclins, cyclin-dependent kinase inhibitors of protein kinase (cdk) proteins responsible for the initiation of DNA synthesis, or too precise chromatid segregation during mitosis, as well as a plurality of proteins encoded by proto-oncogenes and tumor suppressor genes, whose action is also extremely important for the correct cell cycle progression.
Ligase SCF runs from mid-G1 phase through the S phase by mid-G2 phase. In human cells, it ubikwitynuje phase cyclin g! (cyclin D and E) and the cyclin-dependent protein kinase inhibitors (especially p27, p21) and the transcription factor E2F-1 regulates the expression of genes encoding proteins necessary for the start of DNA synthesis in cells or Cdc6 protein (DNA replication factor) binding to transcriptional start site. The basic subunits ligase SCF are: protein Skp1, kullina (platform, which comes to the interaction of individual subunits), one of the proteins of the F-box motif responsible for the recognition and binding of the protein having the subject ubikwitacji and protein Rbx1/Hit 1/Roc 1 characterized by the presence of the RING-finger motif and mediating interactions with the enzyme E2 cullin. Most of the protein substrate before the ubiquitination ligase SCF is phosphorylated by different kinases.

The last step of ubiquitination of proteins directly regulating cell cycle progression carry two multi-component complexes ubiquitin ligases - SCF (Skp1-Cullin-FBP) and APC / C (Anaphase-Promoting-Complex-Cyclosome).
Ligase APC / C is active from mid- G2 phase , in mitosis and early G1 phase . It includes primarily ubiquitin mitotic cyclins ( cyclin A and B, the regulatory subunits of CDK1 and CDK2 kinase operating at mitosis ) , the so-called . sekuryn , which are inhibitors of protein and anaphase chromatid separation , to their regulatory proteins (e.g., Cdc20 ) Polo/dc5 kinase phosphorylation of Cdc25 phosphatase, among others, as well as APC subunits , as well as protein binding Ase with microtubules of the mitotic spindle or start regulating gemininy DNA synthesis in S phase by inhibition of the formation prereplikacyjnego complex (pre -RC ) . Solid components of the complex APC / C (composed of several proteins) are the APC protein and APC11 @ homologous protein of kulliną and Rbx1/Roc1 in this already ligazie SCF . APC2 protein is a basic unit , which attach to other subunit APC11 protein ( the RING -finger motif ) and mediates , is believed to interact with a suitable enzyme E2.
Adjusting the operation of the APC ligase occurs primarily by attaching to specific complex regulatory subunits - proteins or protein Cdc20/Fizzy Cdh1/Hct1/Fizzy-related involved , like protein F -box motif in the recognition and binding of protein substrates . Both the complex of phosphorylated APC / C , and the regulatory subunits is essential for the operation ligase APC / C. Phosphorylation of cdk kinase make active in mitosis , Polo kinase , as well as kinase A, which can both activate phosphorylation and inhibit the action of the ligase APC / C. Moreover, the ligase APC / C is also influenced by the protein of the mitotic spindle checkpoint which are responsible for the precise control of the regularity of the distribution chromatids course mitosis anaphase and termination . Proteins of the BUB and MAD family produce, upon activation of the spindle checkpoint , transient complexes with protein Cdc20 , ligase activity regulator APC / C , thereby inactivating effect and retaining the cells in metaphase . In recent years, have learned the mechanism responsible for the inactivation of the ligase APC / C in interphase . Indeed Emi1/Rca1 identified protein ligase acting as an inhibitor of APC / C , which regulates the synthesis of the transcription factor E2F , and ubiquitination precedes the degradation performed by ligase SCF .
Ligase proteins are substrates of the APC / C is characterized by the presence of the so-called normally . destructive box (D -box) , an amino acid sequence first described in the mitotic cyklinach necessary for substrate recognition by a ligase and a suitable enzyme E2. Mutations in boxing destructive or absence of the amino acid sequence of proteins induce stabilization and retention processes in which they participate. It also appears that the KEN sequence found in some proteins may also be necessary to identify and ligase ubiquitination by APC / C regulatory protein complexed with CDH1 .
Recently it has been shown that the ubiquitin ligase is also active in the protein Chfr checkpoint occurring in mitotic prophase chromosome condensation and monitoring centrosomes and movement towards the poles of the cell. They are characterized by the presence of the FHA domain ( Forkhead -associated ) and RING -finger motif , found the proteins Rbx1/Roc1 and APC11 discussed earlier ligases . Chfr ligase catalyzes in vitro ubiquitination his own , and especially ubikwitynuje and directs the path of proteolysis Polo1 kinase ( Plk1 ), which is involved in the regulation cycle is becoming more and more appreciated and understood. This is because it is largely responsible for the proper phosphorylation state , and thus the activity , phosphatase Cdc25 , and cdk activating kinase kinase wee, which inhibits them . The result of the reduced level of Plk1 kinase activation is delayed Cdc25 phosphatase and kinase inactivation wee, which resulted in disruption of processes that involve kinase cdk before metaphase . It should however be borne in mind that in addition to Plk1 kinase and other kinases phosphorylate these enzymes. Both Cdc25 phosphatase and kinase wee are degraded in the ubiquitin-proteasome system , and their ubiquitination ligase SCF performs likely .
RING -finger motif is also found in the protein MDM2 , which is the ligase ubikwitynującą p53 suppressor protein , then degraded by the 26S proteasome . The intracellular level of MDM2 is tightly controlled at the transcriptional level , and very effectively by complexing with a variety of cellular and viral proteins . Mutants p53 and p73 proteins related p53 and p63 are not recognized and ubiquitinated by MDM2 ligase . The possibility that a complex of p53 - MDM2 determines intracellular localization of these proteins and phosphorylation in each specific amino acid residues. The second , recently identified substrate ligase MDM2 , proved to be the androgen receptor . Its ubiquitination by MDM2 ligase is largely dependent on the activity of Akt. Although either p53 or the androgen receptor is not involved directly in the various stages of the cycle , their precisely controlled proteolysis is extremely important due to the effect of these transcription factors on the expression of many proteins directly involved in the regulation of cell cycle progression , and apoptosis.
Degraded by the 26S proteasome are also membrane receptors (tyrosine kinases) a plurality of growth factors such as FGF, PDGF or HGF. Their ubiquitination ligase Cbl made ​​which acts through the RING-finger motif of the corresponding protein E2 ubiquitin transfer.
In addition to those proteins are also many other proteins important for the course of the cycle is proteolytically degraded, and not only with the involvement of the ubiquitin-proteasome system. Examples of such proteins are c-myc and pRb, both degraded in the proteasome (ubiquitin ligase hitherto unknown) and the calcium ion-dependent cysteine ​​protease - calpain (c-Myc) and caspase (pRb).
In this paper is focused on discussing the ubiquitination of proteins regulating the cell cycle. Be aware that it is equally important , though less known is the process of deubikwitynacji in situations of improper labeling of ubiquitin . Carry him numerous (> 90 previously known ) cysteine ​​proteases , whose role in the regulation of the cell cycle is now beginning to be appreciated.

As seen, the cycle regulators protein proteolysis of many proteins involved . You should be aware that even their intracellular levels may vary due to various factors, which may result in undesirable or at any given time to stabilize regulatory protein , or its accelerated decomposition . As already known , abnormal protein ubiquitination and deubikwitynacji underlie many diseases , especially proliferative , autoimmune and neurological disorders. The current state of knowledge about the course of the basic pathways of ubiquitination and degradation of proteins makes in recent years may have already started research focused on the chemical control of proteolysis cycle regulatory proteins .

Author of the article: 

The Editorial Board
Andrzej Łukaszyk - przewodniczący, Zofia Bielańska-Osuchowska, Szczepan Biliński, Mieczysław Chorąży, Aleksander Koj, Włodzimierz Korochoda, Leszek Kuźnicki, Aleksandra Stojałowska, Lech Wojtczak

Editorial address:
Katedra i Zakład Histologii i Embriologii Uniwersytetu Medycznego w Poznaniu, ul. Święcickiego 6, 60-781 Poznań, tel. +48 61 8546453, fax. +48 61 8546440, email: mnowicki@ump.edu.pl

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