Mutation in a single cells causes cancer over a period of time in a person’s life.
The genes that play a role in causing cancer are:
a) tumor suppressor genes
b) proto oncogenes
Mos is a proto oncogene that is responsible for coding the serine/threonine kinase.
In 1980 ,the transforming gene of Moloney murine sarcoma virus (v-mos) which causes cellular transformation was identified to be Mos. One of the first molecularly cloned proto oncogene was Mos. It was identified that when c-mos gene is abnormally expressed it induces oncogenic transformation of somatic cells.
The transforming activity of mos depends on its kinase activity. It was concluded that the expression pattern was restricted to “germ cells” even though it is an oncoprotein.
In response to a variety of extra cellular growth factor-receptor interactions at the cell surface, the mitogen –activated protein kinase (MAP kinase/MAPK) also called extracellular signal – regulated kinase (ERK) is activated which leads to the transcriptional activation of immediate early genes.
In oocytes ,MAPK is activated independently of growth factors and tyrosine kinase receptors,acts independently of transcriptional regulation , contrals the G2-M period of the cell cycle and its under the control of the regulator Mos.
The expression of Mos protein is restricted when compared to its transcript,specific to time and place.the enzyme gets accumulated during oocyte maturation and undergoes selective proteolysis upon fertilization.
At first, the physiological meiotic functions of mos appears to be different based on the oncogenic properties of the kinase in the somatic cells.ovarian teratomas are developed by parthenogenesis as result of loss of mos in mouse,which establishes its activity as oncoprotein in somatic cells and which makes it ceratin mos could be considered as meiotic tumor suppressor gene. This is conflicting to its ability to induce M-phase entry of oocytes,to seize mitotic cleavage of Xenopus unfertilized oocyte and its transformation ability of mammalian fibroblast. Queries are raised as we observe the function of Mos in female germ cells which appears to be largely mediated by MAPK. The equivalent of proto oncogene Raf-1 in animal oocytes is mos ,a MAPKKK. Although Raf-1 is expressed in germ cells , mos selective to activate the MAPK cascade during meiotic division.
More about Mos kinase ,its activity, the methods for analysis in various model organisms like mice,frog and its contrast with human gene is discussed in this paper.
1.2Role in oocyte meiosis:
The large size and the ease of manipulation in the laboratory makes vertebrate oocytes particularly useful models for research on cell cycle. the discovery and the purification of maturation promoting factor (MPF) from frog oocytes. Like any other species ,the meiosis of frog oocytes is also regulated at two unique points in the cell cycle,and further studies of oocytes have cast light upon the mechanism of cell cycle control.
Diplotene stage is the first regulatory point in the oocyte meiosis of the first meiotic division. In humans,the oocytes can remain arrested for long periods of time at the diplotene stage ,almost 40-50 years.the arrest during the diplotene stage causes decondensation of the oocyte chromosomes ,which are actively transcribed. As a result of this transcriptional activity,the oocyte undergoes tremendrous growth during this stage. Human occytes are about 100μm in diameter which is more than a hundred times the volume of a normal somatic cell. Frog oocyte are larger ,with the diameter approximately 1mm. during this stage of cell growth ,the materials that are required to support the development of the embryo are accumulated,including RNAs and proteins.the early embryonic cell cycles take place in the absence of cell growth, rapidly dividing the fertilized egg into smaller cells.
The timing at which meiosis resumes and fertilization occurs in oocytes varies from species to species.the oocytes remain arrested at the diplotene stage in some animals,and then later it proceeds to complete meiosis.in oocytes of most vertebrates including frogs,mice and humans ,meiosis resumes in response to hormonal stimulation and proceed through meiosis I before fertilization. Meiosis I following cell division is asymmetric ,which results in the formation of small polar body and an oocyte that retains its large size. In the next step , the oocyte progresses into meiosis II without the nucleus being re-formed and the chromosomes are decondensed.
The second point of control,where the control by mos kinase takes place is at the metaphase II where the cell division is arrested,and it remain in this stage until fertilization.
The meiosis of oocytes is controlled by MPF ,similar to the M phase in somatic cells. The MPF regulation displays unique features that are responsible for metaphase II arrest. As at the G2 to M transition of somatic cells, the resumption of meiosis by activating the MPF is initially triggered by hormonal stimulation of diplotene –arrested oocytes .MPF initiates chromosome condensation ,nuclear envelope disintegration and the formation of spindles .
The anaphase –promoting complex B is activated that causes the transition of meiosis I,from metaphase to anaphase,which is accompanied by the decrease in the activity of MPF. After cytokinesis ,MPF activity rises and it remains high while the oocyte is arrested at Metaphase II. A special regulatory mechanism is found especially in oocytes which controls the MPF activity during metaphase II, preventing the transition from metaphase to anaphase in the meiosis II and the cyclin B proteolysis during a normal M phase by the inactivation of MPF.
MOS has been identified as a protein –serine /threonine kinases to be and essential component of CSF using various experiments. Mos is specially produced in oocytes after the completion of meiosis I and is required for increase in MPF activity during meiosis II and also for the maintanence of MPF during metaphase II. The activation of ERK MAP kinase which plays an important role in cell signaling pathways results in the action of MOS . ERK plays a different role in oocytes as it activates another protein kinase called Rsk ,which prevents the activity of anaphase-promoting complex and meiosis seizes at metaphase II. This enables the oocytes to remain dormant at this point in the meiotic cell cycle for several days ,awaiting feritilization.
Figure 1: Progression through meiosis and timing of fertilization: what does Mos do? Oocytes are arrested at prophase of the first meiotic division (prophase I). Under response to a physiological stimulus, MPF is activated and promotes breakdown of the nuclear envelope (GVBD for germinal vesicle breakdown) and formation of the metaphase I spindle. In insects, molluscs, and ascidians, oocytes arrest at metaphase I until fertilization. In the other cases, oocytes extrude the first polar body and enter the second meiotic division. In vertebrates, they arrest at metaphase II until fertilization. In echinoderms and cnidarians, they complete the second meiotic division by emitting the second polar boy, reform a nucleus (female pronucleus), and stop at the G1 phase of the first cell cycle until fertilization. In different species including the nematode Caenorhabditis elegans, fertilization occurs at prophase I and corresponds to the stimulus promoting meiotic maturation. Mos has been implicated: (i) in the initial step of MPF activation during reinitiation of meiotic division, (ii) during the metaphase I to metaphase II transition for the suppression of S-phase, for the microtubular spindle organization and for the reactivation of MPF to enter meiosis II, and (iii) in the arrest of oocyte maturation before fertilization.
2.2 ADVANCES IN MICE:
In mouse oocytes, Mos is an upstream activator of mitogen activated protein kinase (MAPK) and is responsible for the arrest at metaphase II. Female mouse that are mos deficient (mos –
/-) undergo parthenogenesis activation and the oocytes derived from these mice fail to are arrest the cell cycle at metaphaseII and are less fertile.
The first meiotic division of mos -/- oocytes phenotypically resembles mitotic cleavage or produces abnormal large polar body. In these oocytes the shape of the spindle is altered and fails to translocate to the cortex,which leads to the establishment of an altered cleavage plane. In these oocytes the polar body persists instead of degrading and sometimes even undergoes additional cleavage ,which in turn provides conditions for parthenogensis.
The oocytes are fixed with 1.8% paraformaldehyde in PBS for 40 min at room temperature,and 1 %Triton X-100 in PBS for 20min is added to permeabilize after removing the zona pellucid with acidic tyrode solution at Ph 2.5. the oocytes are washed with 0.1% Tween 20 in PBS for 20 min and incubated with PBS containing 3% BSA ,10% goat serum and 0.1% Tween 20 which is used as a blocking solution for 1hr at 37ᵒc. Using anti-tubulin antibody ( YL ½ /2 ,accurate chemicals ) at a 1:40 dilution the oocytes are incubated and the again fluorescein isothiocyanate conjugate secondary antibody at 1:20 dilution.incubation was performed at 1 hr at 37ᵒC for both secondary and primary antibodies . co staining was performed with 4´,6-diamidinophenylindole (DAPI) for visualization of DNA. Zeiss 310 confocal laser scanning microscope was used to examine the immunostained oocytes.
Based on the MAPK activity,the results observed were,
Mos is a potent activator of the MAPK pathway , we asked whether MAPK was activated in the MOS-/- oocytes. Normally, after removal of IBMX, MAPK activity is detected as early as 5 hr after initiation of meiotic maturation and remains high throughout maturation. Extracts prepared from oocytes at the GV which are immature oocytes, Metaphase I which are obtained 7 hr after meiotic initiation and Metaphase II obtained 14 hr after meiotic initiation stages were tested for MAPK activity by using myelin basic protein as a substrate. In MOS+/+oocytes, high MAPK activity was detected in both Metaphase I and Metaphase II oocytes . In contrast, MAPK activity was not detected in the stage Metaphase I or Metaphase II MOS-/- oocytes . Thus, MAPK activation during mouse oocyte maturation depends entirely on the presence of Mos.
During meiotic maturation, In Mos +/+ oocytes high MAPK activity was detected in both MI and MII oocytes. In contrast ,MAPK activity was not detected in MI or MII Mos -/- oocytes.
ABNORMAL FORMATION OF FIRST POLAR BODY:
Mos -/- oocytes were examined for possible phenotypic alterations during oocyte maturation .
There was no difference observed between mos -/- and mos+/+ during germinal vesicle breakdown (GVBD).However dramatic abnormalities in the formation of first polar bodies were observed in mos-/- compared to mos +/+. Under normal conditions ,at the metaphase I the spindle is centrally positioned in the oocyte and then migrates to the cortex. This is necessary for the asymmetric cleavage that produces small,15-20μm in diameter polar body and the remaining larger competent secondary oocyte. We observed that 20-30% of the mos-/- oocytes extruded abnormally large polar bodies ;half were 30μm in diameter while the remaining half underwent mitotic like cleavage producing cells of equal size at the end of the metaphase I.The anti-tubulin and DAPI staining revealed these abnormal cell divisions correlated with the failure of the metaphase spindle to properly translocate to the cell surface. Hence in the absence of Mos/MAPK , 20-30% lose their ability to properly position the spindle apparatus. Despite abnormal cytokinesis ,most of the oocytes proceed to metaphase II. However we are not sure of their competence for fertiliziation or embryogenesis although mos -/- females showed reduced fertility. Nucleus reforms in 90% in these oocytes by 24 hr.
These results are in concordance to the results that Mos/MAPK influences the positioning of spindle in the metaphase during meiosis and are also consistent with the results that were identified that Mos and MAPK are involved in microtubule reorganization.
In the normal oocyte,the microtubule arrays present during prophase disappear as the spindle apparatus begins to form at 5 hr after meiotic initiation. Anti-tubulin antibody immunostaining showed that in mos -/- oocytes the microtubule array persists through metaphase I and even after 7hr after meiotic initiation. As MAPK is held responsible for severing activity of microtubules arrays and hence in the absence of Mos/MAPK the extensive astral like microtubule arrays seem to persist and thereby prevents the proper association of the spindle with the inner surface of the oocyte cortex . these results identify that spindle positioning is a new important Mos/MAPK function and consistent with the observation that the expression of Mos/MAPK in the somatic cells leads to anastral meiotic –like spindles positioned adjacent to the cell membrane that give rise to binucleated cells.
FIG. 2. Abnormal positioning of spindle apparatus and first polar body formation in MOS-1- oocytes. Oocytes were selected before (at 8 hr) (a-c) and after (9 hr) (d-f) extrusion of the first polar body and stained with anti-tubulin YL½/2 antibody (green) and DAPI (red). In control MOS+/+ oocytes, one spindle pole is juxtapositioned to the membrane (a) and a normal-size polar body is extruded (d). In contrast, in MOS-‘- oocytes, the spindle apparatus is observed in the center rather than at the membrane (b, c, e, andf); a large polar body (b and e) and symmetric cell division (c and f) are also shown.
Expression of MOS kniase in somatic cells:
In maturing Mos-/- oocytes it was observed that in 90% of the oocytes there was long term persistence of the first polar body. In mos +/+ ,after extrusion the first polar body is degraded by 7hr ,while in mos-/- oocyte the persist for more than 11hrs. not only do the polar bodies persist ,but they also elongate and frequently undergo an additional division .
The life span of the first polar body in mos-/- oocytes suggests that rapid degeneration of the first polar body may be a programmed event that is triggered by Mos/MAPK .It was observed that mos/MAPK trigger apoptosis in somatic cells. The mos/MAPK is involved in the degeneration of the first polar body might explain the significantly longer life span of second polar body , which extrudes after fertilization when mos/MAPK are being degraded and inactivated.
Hence, it can also be concluded that another unique mos/MAPK is to cause programmed degradationof the first polar body which explains one form of parthenogenesis. This parthenogenetically activated mos -/- oocytes in the follicle before ovulation raises the possibility that they will cause abnormal meiotic division.
A similar situation can be seen in the oocytes of LT/Sv mutant female mice ,in which we can observe the arrest and ovulate at metaphase I and II and spontaneously undergo parthenogenetic activation in the ovary.
Fertilization of the primary oocyte from LS/Sv mutant female mice is possible.there is no assurance that the persisting large polar bodies of mos-/- oocytes are competent for fertilization ,but the diploid ovum resulting from an error is fertilized in the first meiotic division and has been proposed as one mechanism responsible for combines placental mosaicism(CPM). In the event of high conceptuses(1-3%) CPM occurs in high percentage and it may be attributed to the acquisition of an extra haploid set of maternal (digynic) chromosomes.
An alternative mechanism for CPM can be provided due to the loss of mos/MAPK function and the long term stability of polar bodies in mos-/- mice . a prerequisite for the continuous genetic integrity of the species is the accuracy of chromosome partitioning during oocyte meiotic maturation. As expected the alteration observed in mos-/- oocytes leads ro increases genetic instability ;just as proposed the inappropriate expression of mos/MAPK in somatic cells increases genetic stability due to aberrant expression of meiotic activities
The first studies on physiological function of mos were conducted on Xenopus oocytes and it was found that GVBD and MPF were inhibited when mos antisense oligonucleotides was injected and that the injection of mos RNA activated MPF and induced GVBD in the absence of progesterone.the period for protein synthesis is necessary for the MPF activity in frog oocyte whe compared to mouse or starfish. It was reported by Yew er al. that mos protein efficiently induces GVBD and the activation of MPF I the absence of protein synthesis but in the presence of low concentrations of progesterone unable to trigger meiotic maturation. that has lead to the conclusion that mos is the only synthesized protein required for initiating maturation. The other constitutively active downstream effectors that are also able to induce meiotic maturation when injected into the oocytes are Mos,MEK,MAPK and p90rsk . the MPF activation under effect of mos is mediated through MEK/MAPK/p90rsk as mos is inactive when microinjected due to the presence to pharmacological MEK inhibitor,U0126. On the whole, MPF activiation is a result of a linear chain of molecular events initiated by progesterone,which starts with the synthesis of mos protein followed by subsequent activation of the MEK/MAPK/p90rsk cascade that eventually leads to the control of Cdk1 catalytic subunit of MPF ,Myt1 kinase that phosphorylates and inactivates Cdk1 and the Cdc25 phosphatase that specifically activates Cdk1.
However, this view was then questioned by several studies. Gross et al. and Fisher et al. showed that progesterone is able to activate MPF by a mechanism independent of MAPK . this result is difficult to reconcile with the requirement for mos downstream of progesterone in Xenopus oocytes. When considering the idea that MAPK activation downstream of mos synthesis is not required for maturation ,the inhibition process of mos synthesis by morpholino antisense oligonucleotide does not succeed to block the progesterone –stimulated GVBD. This conflict between the requirement of mos/MAPK cascade to activate MPF in frog oocyte was recently reconciled. It was revealed that MPF activation induced by progesterone injection is completely invalid when the cyclin B synthesis and mos/MAPK pathway are simultaneously impaired. The recovery of at least one pathway restores MPF activation. This demonstrates that MPF activation requires either mos/MAPK pathway or cyclin B synthesis. In contradiction to cyclin B accumulation caused by progesterone independently of MPF activation ,the accumulation of mos requires a stabilizing phosphorylation catalyzed by MPF and as a result MAPK activation only happens when MPF activation is already initiated. This difference in regulation in accumulation of mos and cyclin B1 suggests that the physiological pathway induced by progesterone depends on cyclin B synthesis and mos/MAPK cascade contributes to the MPF activation only after mos stabilization is obtained. Even when cyclin B synthesis is impaired some other rescue mechanism recruits the mos/MAPK pathway,which allows it to complement the deficiency in cyclin B. this explains the phenomenon that in organisms such as jellyfish,starfish even when mos is not expressed yet MPF is activated,hence the mechanism of initiation by cyclin B and mos/MAPK pathway was analysed.
3.2 Suppression of DNA replication:
In Xenopus, during maturation the ability to replicate DNA was acquired at the beginning of meiosis I by Cdc6,the synthesis of the only missing replication factor ,which is necessary for recruiting the minichromosome maintenance (MCS) helicase to he prereplication complex.
After the germinal vesicle breakdown ,the maturing oocyte is fully equipped with the replication machinery that prevents the entry into S-phase until fertilization. In frog oocyte ,mos is required during metaphase I to metphaseII transition to suppress the S-phase.when the synthesis or the activity of mos is specifically inhibited or when MAPK activation is prevented, frog oocytes completes meiosis I but nuclear envelope reforms and DNA replication occurs. successfully similar results have been obtained in starfish oocytes. However the results obtained from mouse oocytes differ,concerning the involvement of mos and MAPK in the Sphase suppression and the entry in to meiosis II. The alteration of mos by antisense oligonucleotides either arrests oocytes before the extrusiom of first polar body or induces nuclear reformation and DNA replication after meiosis II in Xenopus. In contradiction ,oocytes from mos gene knockout mice enter meiosis normally,despite interphase like microtubular stage. the reasons they contradict might be due differences in the strains of mice or the experimental strategy used for deletion. In jelly fish oocytes mos abalated by morpholino antisense or MAPK activation prevented by U0126,and still GVBD occurs on time ,but the oocytes fail to emit both first and second polar bodies,however they do not form a replicating nucleus. This demonstrates that the ability of mos to suppress DNA replication between two meiotic divisions is not a universally present function. These differences are related to the presence or absence of functional replicative machinery in oocytes depending on the species.
Until now, the molecular mechanism controlled by the Mos/MAPK cascade and leading to the prevention of DNA replication is not fully been discovered. All the findings firmly suggests the critical function of Mos at meiosis I-meiosis II transition in vertebrates: the Mos/MAPK module is involved in MPF reactivation that depends both on the arrest of cyclin B degradation, initiated at the exit of meiosis I, and on new cyclin B synthesis, allowing MPF reactivation and entry into meiosis II . By controlling this cyclin B turn-over, Mos allows MPF activation and entry into meiosis II. The Mos/MAPK module indirectly controls the replication machinery through the control of MPF activity. Though it was similarly unsuccessful in the meiosis I/meiosis II transition occurred in Xenopus oocytes that were injected either by antisense against Mos mRNA or dominant-negative Cdk1 kinase , it was suggested that MPF reactivation occurring under the control of Mos/MAPK after meiosis I would suppress DNA replication. However, when the reactivation of MPF at meiosis II is prevented specifically by antisense oligonucleotides against B-cyclins, the Xenopus oocytes degrade and , fail to form a second meiotic spindle, and it does not support nuclear organization and DNA replication . This careful analysis favors the view that the Mos/MAPK pathway acts directly to suppress DNA replication, independently of MPF activity.
An unknown capacity for Mos uncovered by perceptions in the oocytes of starfish, mouse, Xenopus, and the jellyfish is its association in the control of axle development and situating and the chromatin association. This was first uncovered by examination of mouse oocytes . Strikingly in mos−/− oocytes or in oocytes where MEK is repressed, the microtubules and chromosomes advance towards an interphase-like state amid the change between two meiotic M-stages and afterward show monopolar half-shafts .After this stage , comparative perceptions were performed in different frameworks . This closely monitored part of the Mos/MAPK in the balance of microtubular cytoskeleton to guarantee meiotic axle development and situating could add to its cytostatic action autonomously on the control of MPF in oocytes captured at metaphase I (as Drosophila) or metaphase II (in mouse). It could also add to the chromosome unsteadiness of tumor cells where mos is upregulated.
3.3 Metaphase arrest in vertebrates:
In vertebrates, the Mos/MAPK pathway serves to settle MPF, guaranteeing a capture at the metaphase arrangement. Discharging this hindrance requires the movement of the APC/C protein complex, a ubiquitin ligase that objectives cyclin B for obliteration . In mouse and Xenopus metaphase II-captured oocytes, APC/C is specifically restrained by the Erp1/Emi2 protein . among oocyte development, Erp1/Emi2 seems simply after metaphase I, clarifying why the oocyte does not stop at metaphase I, but rather at metaphase II . Upon treatment, a transient ascent in free intracellular calcium enacts calmodulin-subordinate protein kinase II that phosphorylates Erp1/Emi2, along these lines making a docking site for the Polo kinase. The Erp1/Emi2 phosphorylation by Polo kinase targets it to obliteration, discharging APC/C from restraint . As a result, cyclin B is debased, MPF movement is in this manner inactivated and the prepared oocyte exits metaphase II. Given that Erp1/Emi2 would itself be able to repress APC/C and balance out MPF, why is the Mos/MEK/MAPK/p90Rsk required for metaphase II capture? Late works gave promising insights about the connections amongst Mos and Erp1/Emi2. In Xenopus metaphase II-captured oocytes, Erp1/Emi2 is a substrate of p90Rsk, and Mos-subordinate phosphorylation of Erp1/Emi2 by p90Rsk is vital for both settling Erp1/Emi2 and building up CSF capture in meiosis II oocytes . All the more definitely, the Rsk-interceded phosphorylation of Erp1/Emi2 advances its collaboration with the protein phosphatase PP2A. PP2A dephosphorylates two unmistakable bunches of buildups in Erp1/Emi2, one in charge of adjusting its steadiness amid the metaphase II-capture and one controlling its official to the APC/C . In this way, Mos and Erp1/Emi2 cooperatively build up and keep up metaphase II capture in Xenopus oocytes .
Figure 3: Meiotic arrest of the unfertilized oocyte: the downstream effectors of Mos/MAPK. In all species, oocytes halt meiosis to prevent embryonic development in the absence of fertilization. Depending on species, meiosis arrests at prophase I, metaphase I, metaphase II, or G1 following meiosis. Except in C. elegans, Mos was found to be the ubiquitous cytostatic factor responsible for the unfertilized oocyte arrest. Its downstream targets accounting for the meiotic arrest of the unfertilized oocytes are indicated.
In mouse, the APC/C Erp1/Emi2 likewise assumes a basic part to maintain the metaphase II-capture of the unfertilized oocyte . Hence, it is sensible to foresee that the component of metaphase II-capture foundation is additionally saved amongst frog and mouse. Be that as it may, this isn’t so. Shockingly, despite the fact that it is settled that the Mos and MAPK are basic for building up this capture, p90Rsk, which is initiated by MAPK as in alternate species , isn’t engaged with the metaphase II-capture of mouse oocytes. This is in solid appear differently in relation to Xenopus or starfish unfertilized oocytes, where it is the fundamental go between of Mos cytostatic action (see earlier and underneath). At that point, the downstream effector of MAPK controlling APC/C through Erp1/Emi2 as well as balancing out the microtubular shaft still requires to be illustrated in mouse.
CHAPTER 4 : EXPERIMENATION ASSAYS:
4.1 IMMUNOPRECIPITAION TECHNIQUE:
Activation of malE-mos protein:
E.coli was used to obtain non contaminated recombinant malE-mos protein,which is a fusion protein and when it is incubated with the extract attained from Xenopus which are extracts that are cell free,the protein gets activated.
In order for us to get cells in the interphase stage we had to treat cells that were seized by CSF with Cacl2 at 24oc for 60 mins after which we treat it with ethylene diaminetetraacetic acid(EDTA) for 5min.
On the whole we used 1pg of non contaminated malE- mos protein which was placed in the interphase solution of cells for 60 mins at 24oc.
The obtained product was diluted to 10x with the kinase buffer which was a mixture of B-glcerophosphate, magnesium chloride and EDTA, which had all been maintained at very low temperature. The above process results in activated malE- mos protein which can be immunoprecipitated when placed in anti-mal/E anitibody on ice for 3 hrs.the obtained precipitates were got back by maintaining it with protein A at ice cold temperature for half hr and it is washed multiple times using a buffer whose composition is Tris-Hcl, ph maintained at 7.5.sodium chloride, Tween and many chemicals that can prevent the activity of proteases.
Phosphorylation of MAPKK :
From rabbit skeletal muscle, highly concentrated MAPKK is obtained and activated using preotein phosphatase 2A for 30mins.
Using okadaic acid the activity of the phosphatase enzyme was prevented. in order for us to get the kinase reactivity, the precipitates obtained were washed with a list of chemicals such as Tris-cl, EDTA, mercaptoethanol and briji-35 and then incubated with the non reactive MAPKK and magnesium acetate.
The phoshorylation activity were studied using SDS-PAGE and autoradiography.
Activation of MAPKK in the lab:
The precipitates of malE-mos obtained were washed with TrisHCl , EDTA, mercaptoethanol, briji-35 and then placed in final volume of 200PM ATP, magnesium acetate,inactive isoform of MAPK from E.coli in the absence or presence of MAPKK obtained from PC-12 cells that are non stimulated.
We can also use MAPKK obtained from E.coli as well. The supernatant is obtained from the above mixture,after placing it in 3oc for half hour,and then it was diluted 5x in cold TrisHCl, EDTA, 1ml BSA, and using myelin base protein (MBP) assay was carried out for MAPK.
Activation of MAP kinase in mouse fibroblasts expressing mos:
Since we need a cell line that has the Xenopus mos gene ,we use NIH 3T3 cells.
The cells undergo cotransfection with MLV-Mos and pMEXneo.we used the process of calcium and phosphate precipitation.we also created cell line that is used as control, in which only tranfection of MLV-mos and pMEXneo was done.
Results of the experiment:
The working of mos kinase can be analysed by using the malE – mos protein that is obtained by using recombinant techniques and the enzyme is not active when it is extracted from bacteria and then it is activated with a mechanism which is not clear yet, using the extracts obtained from Xenopus oocytes and they lack cells in them. For the apparatus to work, we utilize extracts which are arrested in the interphase,to obtain purified samples to prevent the presence of cyclin b in MPF, when we perform immunoprecipitation. In order for us to identify autophosphorylation,we made use of working malE – mos protein which was obtained from the previous process with antibodies.
As we already know the working of mos kinase the precipitate obtained, is used in the phosphorylation of MAPKK which using the PP2A treatment was non activated. The working pf mos kinase in the activation of MAPKK ,can be analysed as the precipitates obtained from unstimulated PC-12 cells and recombinant p42mapk ,which can later be further studied using the supernatant for the working of MAPK.From this experiment it can hence be concluded that mos which was obtained by precipitation works as MAP kinase kinase kinase that can activate MAPKK by phosphorylation. It can be explained that mos that is obtained from using recombination brings about activation of extracts obtained from Xenopus. It may be suggested that the activation takes place by addition of phosphate.
Through this experiment we get to know that mos is activated by the addition of phosphate. Even though there are many contradicting views based on the activation by the addition of phosphorylation ,it is mostly accepted that mos is involved in the Xenopus oocyte maturation by addition of phosphate to the serine residues.
Concerns related to the experiment:
• Due to non covalent modifications ,there are suggestions that certain MAPKKK are activated.it was hard to prove this suggestion because the mos protein obtained as a result of immunology and precipitation ,was very heat sensitive as it begins to denature at 30oc even after 6min
• Sometimes it was observed that the okadaic acid used to prevent the addition of phosphate fails to function which leads to us questioning the function of phosphorylation.
• We can still not put a label as to why there is instability of the protein, as proteolysis cannot be considered a reason because the immunoprecipitates are once again activated where we add the cell less extracts from Xenopus.
• Different extracts can be used to activate which makes the process a little uncertain,as we use not only Xenopus but also extracts obtained from PC-12 cells to activate the immunoprecipitated malE – mos protein .recently it was found that even rabbit reticulocyte lysates can be used. The reticulocyte was used to activate MAPKK in the lab environment.
4.2 Mos kinase in somatic cells:
Recent studies have taken interest in analyzing whether mos gene expressed abnormally in the somatic cells can trigger the MAP kinase cascade in the area of interest , since it was already studied to great lengths the expression of mos kinase in oocytes of organisms.
A new assay was developed to fulfill this purpose ,with the main component as the transcription factor Elk-1,found in fibrolasts. this factor was based on continous addition of phosphates at several regions in the C –terminal region.
Same process of stimulation of cells was carried out and extracts contained a 42kDa kinase. For the processing of MAPK in vitro, we use Elk-1 is used as the substrate which os phosphorylated by the kinase obtained from the extracts. The process of co transfection was performed between the cells that contained plasmids lexA/Elk-1 fusion protein and Xenopus mos protein.
The action of mos in triggering addition of phosphorylation in Elk-1 is very identical to products obtained using stimulation for 15 minutes using serum ,or continuous expression of ras kinase that is activated, which is a well studied as the activator of MAPK cascade in fibroblasts of mammals.
The MAPK sites are phosphorylated in the C terminal region of Elk-1 as a result of induction by serum ,in the fibroblasts it was concluded that it was the basis by which the transcription is triggered.
Hence when transcprition was stimulated, it was decided that mos can also activate the MAPK pathway in mammalian fibroblasts by addition of phosphate to Elk-1 which eventually leads to the activation of Elk-1. In order to get solid real life proof, we created a cell line which expressed mos oncogene and was obtained from fibroblasts.
In the absence of using serum ,the cell line with parts of both p42mapk and p44mapk are present which indicates that the phosphorylated forms have low electrophoretical mobility.
In order for us to relate if the phosphorylation of p42mapk and 44mapk are connected on the basis of activity we analyse the substrate of MAPK which is obtained from extracts to phosphorylate MBP.the kinase activity of MBP kinase was more in cells that produce mos kinase rather than the control cells that lack production of mos enzyme. In order to get clear and detailed results we studied the activation of using MBP kinase activity when specially in relation to p42 mapk which showed 4x increase in activity when immunoprecipitated.
The result obtained indicates that mouse can trigger the MAPK cascade in the fibroblast obtain from mouse.This result is concordance with the conclusion obtained before that MAPK that is activated by phosphorylation is important not only in the processes and functioning in the mos protooncogene in the oocytes but it is very important in the transformation process of somatic cells.
The process of cellular transformation of somatic cells by oncogenes is a process that is not elaborately explained and the exact role of MAPK phosphorylation which leads to activation is still unclear.Even though the process is unclear it is not in contradiction to the results that state that unnecessary production of an important , highly needed and preserved component of the signal transduction pathway is not related in the transformation of the somatic cells.
CHAPTER 5 :SCOPE
5.1 IN RELATION TO LUNG CANCER:
Experiment performed in somatic cells gave results that c-mos had negative results on being expressed in the cell cycle which led the conclusion that mos protooncogene might play a persistent role in the determining the instability of the genes and variation in kinetics of cells.
The expression of a gene obtained from moloney murine sarcoma virus in 1980s was identified as v-mos.A cellular homologue of this oncogene is c-mos protooncogene.The serine-threonine kinase activity was encoded by a Mr 39000 protein on the chromosome region 8q11-12.
In the MAPK cascade the semos is used as a positive regulator by adding a phosphate to MAPK kinase.As an element of the cytostatic it plays a regulating function in the maturation of germ cells.It seizes the germ cells at metaphaseII by keeping in control the maturation promoting factor.On the whole the exact formation and assembling of the meiotic spindle is related to the c-mos activity which in turn responsible for the asymmetric division of the germ cell in the formation of first polar body and the secondary oocyte.
C-mos has a well defined role in the germ cell maturation but the detail function and more information about the structure is very little known regarding human somatic cells.In somatic cells such as fibroblast obtained from mouse can be used to analyse the continuous expression of c-mos which leads to the transformation of somatic cells into oncogenic cells.There is solid proof tha c-mos can transform somatic cells to ongenic cells like the other oncogene when it translated in the G1 phase.Many G1 targets such as c-fos, c-jun and c-myc are regulated using MAPK pathway as a result of c-mos expressed as mitogenic stimulus.
The main regulator which plays an important role in the c-mos/MAPK pathway is c-fos that results in the cells being transformed.The very recent experiments proved that when v-mos is used for the cells to be transformed in the absence of serum it led to increased level of cyclins such as D,E and A.They were presence of cyclin dependent kinases such as p33cdk2 and p43cdc2 and S-phase specific E2F complexes that leads to to the conclusion of the inability of the c-fos to lower these important cell cycle controlling molecules which may lead to formation of tumor.It was also found that the role of c-mos in the transformation process was not only in the G1 phase but plays an important role in the M phase as well.
The function of c-mos in the mitosis phase are classified as meiotic like changes that results in the formation of two nucleated cells which suggest the mechanism of CIN.
Modern researchers have identified that the function of c-mos may be based on p53.
The relation between c-mos and p53 in series of 56 non-small cell lung cancer tissues was observed to be :
a)status of c-mos
b)assosiation to the non stable nature of the genes and the two components of kinetic activities relating to tumors proliferation and apoptotic indexes.
c)in relation with p53 modification and their association with the above components.
On analyzing tumors there was 27% over production of c-mos.The amount of expression increased in higher stages such as 2 and 3 to 34% when compared to stage 1 17%.Total similarity has been established between c-mos overexpression and increased c-mos mRNA
Levels.Even though c-mos protooncogene was not detected to be amplified its down regulated expression is due to increased transcription.Most of the c-mos positive cases approx 77% were related to aneuploidy.
In the brain display progressive neuronal degeneration and gliosis seems to occurs in transgenic mice,due to the expression of oncogenic proteins-serine/threonine kinase mos at high levels.there is a dramatic increase in the number of astrocytes positive for gilial fibrillary acidic protein,vimentin and possibly tau which is due to gliosis which develops in parallel with the onset of postnatal transgene expression . vimentin in normally expressed only in neoplastic astrocytes, but they are induced to high levels in mos –transgenic mature astrocytes.mos can activate the mitogen activated protein kinase which has been implicated in alzheimers-type tau phosphorylation.
In the mos transgenic brain,increased phosphorylation is found at one epitope in tau containing sereines 199 and 202,a pattern similar but not identoval to alzheimers disease.
The mos transgenic mice expresses a neurofilament realted protein which might be a proteolytic neurofilament heavy chain degradation product.the cytoskeletal proteins that contribute to neuronal degeneration in neurons is caused due to activation of protein phosphorylation.