Although each ischemia and hypoxia bring about decreased oxygenation, an ischemic insult may possess a alot more significant result as a consequence of the more reduction in provide of nutrients and decreased waste removal. Consequently, ischemia is usually a severe ailment and like in brain and heart, contributes to your pathogenesis of the quantity of retinal disorders . Neighborhood or systemic defects in the circulatory system are standard causes of retinal ischemia with local impairments, mainly occlusion of terminal arteries, contributing more often to pathological ischemia. Just about the most regular arterial ischemic issues of your retina contain central retinal artery occlusion , branch retinal artery occlusion , and amaurosis fugax . Importantly, impairment in the retinal circulation influences the inner retinal layers but doesn’t commonly trigger the outer retina to come to be ischemic since it is metabolically supported by the choriocapillaries. Being a consequence, retinal ischemia in most cases prospects to neuronal damage in the inner retina devoid of strongly affecting photoreceptors . Nevertheless, failure on the choroidal vascular technique or separation on the outer retina from your RPE during retinal detachment, results in outer retinal ischemia.
Full retinal ischemia, affecting each the outer and inner retina, also can occur by occlusion in the ophthalmic artery, which supplies every one of the structures within the orbit . Total occlusion of your arterial blood provide effects in ischemic edema within the inner layers, ultimately top to inner retinal atrophy . In contrast, a partial arteriolar occlusion might result in hemorrhages and microaneurysms in affected parts that Wortmannin cell in vivo in vitro can also be accompanied by retinal edema . While in the case of complete or partial venous outflowocclusion,microaneurysms, hemorrhages, exudates and edema are often observed . Chronic retinal ischemia is generally linked to capillary occlusion in the retinalmidperiphery to periphery. That is frequently the end result of systemic cardiovascular, metabolic or hematological ailments. Retinal capillary non perfusion is typically encountered in different retinopathies and is especially studied in retinal vein thrombosis and PDR 1.
Effects of ischemia and hypoxia on retinal perform Disturbance in the delivery of oxygen and metabolites has inevitably unfavorable effects on cellular metabolic process and could be detrimental to neuronal perform. Electroretinograms from the two humans and animals demonstrate that ischemia and hypoxia mainly have an impact on the b wave, which represents a mixed response of different retinal cell sorts which includes bipolar, amacrine, and M?ller reversible Proteasome inhibitor glia cells . The extent from the reduction in b wave amplitude depends on the duration and severity on the insult and about the degree of recovery .

This outcomes in elevated mitochondria permeability, release of cytochrome c, caspase activation and ultimately apoptotic cell death. Our benefits indicating that phosphorylation of Poor at Thr21 by Epo activated JNK1 drastically lowered the association of Negative with Bcl XL in vitro , thus inhibiting the professional apoptotic action of Poor, is constant with our preceding reports . Given that the surrounding amino acids of Thr21 do not comprise the 1 binding web-site , it is actually conceivable that binding to one proteins is not going to be the sole mechanism for sequestrating Awful within the cytoplasm. Glucokinase and phosphofructokinase one happen to be shown to associate with Poor while in the cytosol and contribute for the cell survival. Even further research are wanted to take a look at if glucokinase or phosphofractokinase 1 participated in decreasing the binding affinity of Negative to Bcl XL and avoiding the translocation of Lousy to the mitochondria in HCD cells.
In conclusion, here we reported that JNK1 activation is required for Epo mediated cell survival. Epo activated JNK1 phosphorylated Terrible at threonine 21, therefore inhibiting Terrible association with Bcl XL. Substitute of threonine 21 by alanine in Bad promoted Epo withdrawal induced apoptosis. Therefore, our results deliver a molecular mechanism by which JNK1 contributes for the 20s Proteasome inhibitor survival of erythroid cells. Reduction of apoptosis signaling resulting from deregulation of Bcl2 household proteins is recognized to play a essential purpose in tumor initiation, progression and resistance against therapy. The key regulators of apoptosis, Bcl2 loved ones proteins are localized at organelle membranes too as in cytosol and typically controls the release of Cyt.C from mitochondria. The obvious localization of Bcl2 and Bak at endoplasmic reticulum suggests the attainable involvement of ER resident Bcl2 family proteins from the modulation of cell death signaling .
Latest observation indicates that ER targeted Bcl2 and Bcl xL reduce cell death induced Sorafenib kinase inhibitor by several different apoptotic stimuli like ER strain inducing agents this kind of as tunicamycin, and thapsigargin . Anti apoptotic Bcl2 household proteins can also be recognized to impart cell survival by maintaining a lower ER calcium level leading to lowered availability of mitochondrial calcium load with the subsequent failure while in the release of Cyt.C in the course of death inducing stimuli . Bcl2 is alsoknownto play a substantial position while in the regulation of autophagy, in which focusing on of Bcl2 at ER seems to play a essential role in comparison to wild type Bcl2 . The function of Bcl2 can also be acknowledged to be regulated by phosphorylation and its cleavage by activated caspase .

The degradation of securin is required on the metaphase to anaphase transition to liberate the lively form of separase, a protease cleaving a subunit of your cohesion complex that holds the sister chromatids together . Thus, an activated spindle checkpoint prevents the onset of anaphase via inhibition of protein proteolysis along with the upkeep of the chromatid cohesion. Although small is acknowledged concerning the molecular mechanism of spindle checkpoint activation, the recruitment within the checkpoint proteins to kinetochores as well as the pursuits within the kinases Bub, BubR and Mps are required to activate the terminal effector protein, Mad, that straight binds to and inhibits the ubiquitin ligase activity within the APC C . Yet, its even now unclear how the lack of microtubule attachment or even the lack of kinetochore stress is translated into an energetic checkpoint signal. The kinetochore primarily based kinesin protein CENP E may possess a function in sensing the attachment of microtubules to kinetochores and could possibly be involved with initiating the checkpoint signal by activating the BubR kinase .
Also, the so named chromosomal pas senger complex comprised with the Aurora B kinase, INCENP, Borealin and survivin may well possess a purpose in activating the spindle checkpoint selectively Maraviroc selleckchem on a lack of kinetochore tension . Failure within the spindle checkpoint effects in premature separation of sister chromatids even during the presence of misaligned chromosomes, which directly offers rise to chromosomal instability , the perpetual attain or reduction of chromosomes or huge elements thereof. This really is connected with aneuploidy, and that is a significant hallmark of human cancer. The fact is, in lots of tumor cells the spindle checkpoint perform is weakened and the checkpoint signal is not really sustained . Consequently, an impaired spindle checkpoint could possibly directly contribute to your generation of chromosomal instability and tumorigenesis in human cancer. Microtubule binding anti cancer medication The rationale for that use of anti cancer medication that inhibit the function of microtubules will be to inhibit standard mitotic progression by interfering together with the typical function on the mitotic spindle.
In actual fact, lots of chemical compounds targeting microtubules, primarily derived from purely natural sources, exert their main mode of action on proliferating tumor cells by a blockade of mitosis, which subsequently prospects for the induction of cell death . Traditionally, microtubule binding medicines are classified into two groups: agents that stabilize microtubules including various taxanes and epothilones and agents that destabilize microtubules Tivozanib selleck including numerous Vinca alkaloids and colchicine. Then again, it is actually now established that every one of these medication at lower, clinically pertinent concentrations in the nanomolar range suppress the dynamics of microtubules rather than transforming the net polymer mass of microtubules .

MCL , which can be Noxa?s physiological target, contains a PEST domain that regulates its degradation by means of the proteasome and it frequently also accumulates in cancer cells exposed to PIs .Then again, if enough anxiety is generated within the cell to produce caspase activation, bortezomib causes proteolytic processing of MCL to form a kD fragment that in fact enhances cell killing . Therefore, the effects of PIs on Noxa and MCL are now getting exploited by combining them with ABT , a BH mimetic that targets BCL and BCL XL but not MCL . Finally, PIs induce stabilization of Bik and cells lacking Bik are refractory to PI induced apoptosis . Importantly, each Noxa and Bik are so named sensitizer BH proteins, which means that they will enhance the Bax and Bak activation that’s induced by activator BH proteins but can not straight activate Bax and or Bak on their very own . Therefore, either direct Bim stabilization or some other cellular stress ought to collaborate with Noxa and Bik to trigger cell death.
In our hands, Noxa accumulation is known as a universal feature of bortezomib induced cell death in human cancer cell lines, whereas accumulation of Bik or Bim seems to be alot more variable and cell typedependent Part of protein toxicity Endoplasmic reticular tension as well as the unfolded protein response Excessive accumulation Motesanib of misfolded or oxidized proteins within the ER Golgi network triggers a coordinated cellular response that initially promotes cell survival but will in the end trigger apoptosis if cytoprotective mechanisms are overwhelmed. In the core is really a defense system referred to as the UPR , which functions to raise expression of protein chaperones to limit protein aggregation, to increase biosynthesis of structural components in the ER and to inhibit protein synthesis to minimize the load around the ER Golgi network. Upstream manage of your UPR is mediated via activation of 3 ER transmembrane proteins, the serine threonine kinases PERK and IRE along with the bZIP transcription aspect, ATF . Even though a detailed understanding with the mechanisms leading to their activation is still emerging, current proof indicates that Grp BiP plays a central role.
Below basal conditions BiP associates with the luminal domains of all 3 proteins, thereby stopping their activation . Then again, in response to an accumulation fesoterodine of misfolded or aggregated proteins, BiP dissociates from PERK, IRE and ATF due to its higher affinity for the aggregates . PERK and IRE homodimerize and grow to be active and release from BiP allows the kD and kD ATF isoforms to translocate from the ER for the Golgi, where they’re proteolytically processed and activated, permitting them to translocate for the nucleus . Amongst ATF?s targets is XBP, another bZIP transcription element. However, the mRNA encoding XBP will not be effectively transcribed and the item is just not a potent transcriptional coactivator.

In mice, double knockout of Ku and PARP success in early embryonic lethality, clearly revealing the necessary biological contribution of PARP to genomic integrity when Ku is absent . Mechanistically, throughout option EJ it appears that the two strands on the double helix are independently processed and joined as two separate single strand break ligation occasions . Parp null mice show elevated sensitivity to complete physique irradiation, elevated IR induced chromatid breaks in bone marrow cells, and improved killing of stem cells in the intestinal epithelium . Parp null MEFs are very delicate to killing by IR and show delayed DSB restore at Gy . A comprehensive analysis of I SceI GFP reporter DSB fix in xrcc ku mutant CHO versus management cells demonstrates a very similar requirement for Ku for productive joining of complementary versus noncomplementary ends despite the fact that fix happens additional gradually in mutant cells . Also xrcc cells continually go through much even more in depth base reduction, but display little necessity for microhomology in advance of ligation . In this review, chemical inhibition of PARP won’t sensitize wild type CHO to killing by IR, but xrcc cells grow to be fold even more sensitive.
Chemical inhibition or siRNA knockdown of PARP in xrcc cells markedly and exclusively inhibits EJ , foremost to the conclusion that PARP facilitates EJ inside the absence of Ku protein and with no a necessity for practical DNA PKcs Purpose of MRN complicated The position from the MRN complicated in error susceptible finish joining is addressed in a number of kinds of research . In plasmid compound screening based transfection assays a patient derived mutation in NBS decreases end joining fold compared with gene complemented manage cells . Mutant cells also demonstrate reduced MMEJ . A examine of MRE knockdown in human HEK cells carrying an intra chromosomal I SceI substrate resulting in complementary ends exhibits no result on conservative error zero cost NHEJ but minimizes tiny deletions fold . In this research the exonuclease action of MRE is partially implicated in its error susceptible function . In a associated research, evidence is presented to assistance the thought that ATM?s exercise suppresses error prone MMEJ .
In a further review by using a dual I SceI website chromosomal substrate resulting in cohesive ends , knockdown of MRE, RAD, or CtIP in human cells modestly minimizes end joining efficiency but amlodipine not the proportion of error susceptible joining occasions . Through the use of xrcc and ku mutant hamster cells, this research shows that chemical inhibition of MRN affects different EJ . Importantly, both the ku mutant and control cells have enhanced killing by IR when MRN is inhibited. By utilizing an ATM inhibitor, the authors conclude that at the very least one element of MRN?s influence on finish joining is independent of ATM and, for this reason, not an indirect impact of MRN?s part in activating ATM.

Another review applying laser microirradiation also concludes that the catalytic activity of SET is required for de novo monomethylation of HK and recruitment of BP at injury web sites . It is noteworthy that ATMS P foci also are unaffected by BBAP knockdown due to the fact BP knockdown does lead to defective ATMS P emphasis formation . These findings suggest that basically the availability of BP, instead of its localization to damage web-sites, is ample for ATMS P emphasis formation BP binding to HK Me at harm websites By means of its tandem Tudor domains , BP binds with high affinity to dimethylated lysine of histone H , that is constitutively existing in chromatin . A BP WA Tudor domain substitution mutation totally abolishes IRinduced BP concentrate formation . Though the lively unmasking of HK Me through damage signaling promotes focusing on BP to DSBs , it will be now obvious that de novo methylation of HK at DSBs also contributes. In HeLa cells ChIP examination at blog certain I SceI induced DSBs displays that HK Me, HK Me, HK Me all increase with the break blog in association using a pronounced accumulation in the WHSC histone methyltransferase, but only the expand in HK Me and HK Me is blocked by WHSC knockdown .
Also, IR induced BP foci co localize with WHSC foci . Knockdown of WHSC increases cell sensitivity to killing by IR, confirming the Proteasome Inhibitor selleck chemicals biological value of WHSC emphasis formation . Knockdown of WHSC also reduces the formation of IR induced BP foci but not foci from the upstream things gHAX, MDC, and RNF . Accumulation of WHSC and HK Me at DSBs involves gHAX and MDC and takes place by an interaction of your BRCT domains of MDC with WHSC on its IR induced phosphorylation at Ser by ATM. Non phosphorylatable WHSC just isn’t recruited to DSBs and will not assistance HK Me accumulation. WHSC knockdown cells reconstituted with the WHSCSA mutant protein display exactly the same enhanced IR sensitivity as knockdown cells. Consequently, these latest findings implicate DSB dependent de novo HK methylation in recruiting BP to broken websites in an ATM dependent manner.
It really is noteworthy the WHSC SMI-4a selleck gene is defective within a developmental syndrome named Wolf Hirschhorn which has neurological and immunological impairment . One examine suggests a large affinity interaction of BP with HK Me , but this uncovering is simply not confirmed . Also, mouse dot null cells, which lack HK Me, present ordinary induction of BP and ATMS P foci by IR . In fission yeast, Crb, that is structurally associated but weakly conserved in contrast to BP , also binds HK Me . Fission and budding yeasts utilize HK or HK chromatin marks, respectively, for recruitment of Crb to DSBs Tp associated regulation of BP and ATM activation BP is right linked to the Tp tumor suppressor and relevant proteins in response to DSBs , and also the stability of Tp is diminished on BP knockdown .

Knockdown of hSSB or INTS subunits also success in G S and G M checkpoint defects , which signifies the importance of SSB complexes throughout interphase. Immunoprecipitation analyses demonstrate that the two hSSB and hSSB reside in separate complexes with all the widespread subunits hSSBIP and INTS , and that is recognized to interact with RNA polymerase and undergo gene amplification in hepatocellular carcinomas . Just as knockdown of hSSB or hSSB confers IR sensitivity, knockdown of INTS and hSSBIP confers modest sensitivity to IR and camptothecin . Knockdown of hSSB or INTS also benefits in the defective RAD target response to IR and diminished exercise in an I SceI dependent GFP reporter assay for HRR . Comprehending the stage in DSB repair at which the SSB complexes act is confounded by conflicting success. IR induced hSSB foci kind swiftly and show co localization but are more persistent; hSSB also remains linked with chromatin longer than gHAX and MRN .
HSSB localizes inside seconds to nuclear regions containing laser microirradiation or even a particle irradiation . In contrast, IRinduced target formation by INTS is witnessed only at later on occasions and is of uncertain significance . Despite the fact that knockdown of INTS impairs hSSB emphasis formation, this result could be explained from the destabilization of hSSB, which, remarkably, appears for being resulting from regulation of hSSB screening compounds selleck chemicals in the transcriptional degree by INTS . Thus, the existence of a hSSB INTS feedback loop in response to DSBS is proposed . The findings from nuclear foci and co localization experiments are occasionally inconsistent, which makes it troublesome to infer specifically when in which the SSB complexes act all through DSB signaling and processing. Co localization of hSSB together with the MRN complex is seen within min after IR publicity ; on this examine neither MRN, MDC, nor CtIP appears to be needed for hSSB concentrate formation, but contradictory results are reported for MRN . Certain knockdown experiments also recommend gif alt=”inhibitor chemical structure”> that: the recruitment of BRCA to broken online sites demands hSSB INTS acting downstream of MDC and RNF ; hSSB and INTS are demanded for DNA finish resection throughout HRR as assessed by RPA target formation and BrdUrd immunofluorescence without the need of denaturation, and by focus formation by MRN and CtIP . Yet again conflicting results are reported . Cells going through hSSB knockdown also demonstrate defective chromatin loading of MRN and RPA too as deficient submit {SMI-4a|purchase SMI-4a selleckchem translational modification of MDC . In this regard, it is noteworthy that an interaction among NBS and INTS is reported . In summary, 1 can speculate the SSB complexes might act through MRN recruitment by MDC and therefore support maximize ATM activation and recruitment .

Espana et al have reported that breast cancer cell lines transfected with the Bcl xL gene display a increased rate of lymph node metastasis compared with all the untransfected cell lines. TRAF is definitely an adaptor protein involved in death receptor mediated non apoptotic signaling and has also been proven to inhibit apoptosis. TRAF prevents apoptosis in PDAC cell lines by means of death receptor mediated activation of NF ?B. In conclusion, we have now demonstrated that TRAIL exerts strong non apoptotic results in pancreatic cancer cells through TRAIL R and that these results are additional enhanced by overexpression of TRAF and Bcl xL. TRAF and Bcl xL are really expressed in PDAC cell lines and pancreatic tumors. Thus, it could be hypothesized that inhibition of TRAF and Bcl xL for you to block the non apoptotic uncomfortable side effects of TRAIL treatment method will boost the TRAIL based mostly therapy of patients with PDAC. Aurora kinases are essential parts within the control of cell cycle progression, spindle formation and exercise of numerous target proteins. Altered expression is implicated in chromosomal instability and tumorigenesis .
Three members of Aurora kinases are expressed in mammalian oocytes: Aurora kinase A Aurora kinase B and Aurora kinase C . However, there is even now restricted information Vismodegib Hedgehog inhibitor on targets and actions of those kinases in oocyte maturation. Decrease eukaryotes, this kind of as yeast, possess just one Aurora kinase . From the increased eukaryotes expressing greater than one kinase, AURKA and AURKB are structurally and functionally several and therefore are involved in distinct diverse cell cycle regulatory routines in mitosis. Thus, they acknowledge unique targets or web-sites on molecules to get phosphorylated and therefore are activated by association or modification by different proteins protein complexes. In contrast, AURKB shares large homology to AURKC while in the C terminal a part of the molecule containing the kinase domain but differs during the N terminal sequence, which might possibly give for differential or redundant routines and regulation.
From studies on overcoming inhibition of cytokinesis arrest in AURKB mutant cells by overexpression of AURKC, it appears the two kinases are presumably functionally redundant for no less than a few of their activities and targets in mitosis . Since a point mutation within the human AURKC gene that leads to expression of a truncated protein while not a kinase domain is responsible for manufacturing of giant headed multiflagellar polyploid spermatozoa and male infertility in people and simply because Bicalutamide AURKC is highly expressed in both the mammalian testis and oocytes, it has been recommended that it could possess specified functions and pursuits in pre or publish meiotic events in gamete formation, for instance chromatin condensation in sperm nuclei .

Aurora B ranges peak in early mitosis then drastically decline at mitotic exit. In vertebrates, this drop is mediated in component by Aurora B ubiquitination through the anaphase advertising complicated , and subsequent degradation from the proteasome . Latest reports have linked the Cdc p AAA ATPase together with the regulation of Aurora B as well as chromosomal passenger complicated. In 1 review, p and its cofactors Npl and Ufd copurified with Survivin isolated from Xenopus egg extracts . Ufd was proven to become expected for Survivin ubiquitination, and for that localization of Survivin and Aurora B to centromeres. Conversely, the deubiquitinating enzyme hFAM was essential to the disassociation of Survivin and Aurora B from anaphase chromosomes . Hence, this research concluded that p Ufd Npl is known as a favourable regulator from the CPC, since it is required to the localization of Survivin and Aurora B to metaphase centromeres . Surprisingly, a current review contradicts these findings, suggesting that p is needed for your disassociation of Aurora B from chromosomes, that is in flip a prerequisite for nuclear envelope reformation on the end of mitosis .
p is needed for mitotic spindle disassembly and nuclear envelope reformation in Xenopus egg extracts . However, inhibition or depletion of Aurora B relieved this PARP Inhibitor requirement, suggesting that Aurora B is a crucial target of p within this pathway . Certainly, p physically interacted with ubiquitinated Aurora B and was required to extract the kinase from chromatin . Chromosome release resulted in a corresponding drop in kinase activity, arguably as a consequence of dissemination in the kinase from activating clusters . Steady findings were uncovered on depletion in the two Cdc p orthologs in C. elegans . cdc . and cdc . resulted in defects in chromosome decondensation and nuclear envelope reassembly, along with the retention from the Aurora B kinase AIR on anaphase chromosomes . On top of that, RNAi of both cdc . or cdc . partially rescued a hypomorphic temperature delicate allele of air , and resulted in a rise during the phosphorylation of histone H, a conserved target on the Aurora B kinases .
The disparate conclusions reached by these research increase numerous inquiries concerning the cellular pathways that handle Aurora B kinase exercise and functions. To elucidate the regulation of your AuroraBkinase in an unbiased trend,weundertook a C. elegans genome wide display for loss of perform suppressors within the very same air allele used in the review described over, air . Whilst we Ruxolitinib selleck did not recover both of your canonical CDC members of the family in our screen, we did get, amongst a handful of reproducible suppressors, amember on the Afg Spaf subfamily of Cdc p AAA ATPases. KG. CDC . is closely linked to yeast Afg and mammalian Spaf, which type a distinct subgroup of AAA ATPases that also involves an uncharacterized Drosophila protein .

Treatment method of Lu cells with brought about a dose dependent inhibition of ERK activation . In contrast, ERK remained phosphorylated during the resistant cells regardless of remedy with substantial doses from the BRAF inhibitor as much as mM, raising the likelihood that ERK activation could be mediated by a kinase besides BRAF . To verify the outcomes obtained with , as well as to find out if ERK activation was dependent on BRAF, we knocked down BRAF making use of shRNA . Short hairpin RNA mediated BRAF knockdown led to inhibition of ERK phosphorylation in Lu parental cells, but had no result on Lu R cells, suggesting that ERK activation is BRAF independent in these cells. We also examined if secondary mutations in Braf may very well be associated with growth of resistance to BRAF inhibitors. Mutational examination of exons and in the BRAF gene was performed in all parental and resistant cell lines. These exons signify people during which mutations in melanoma and genetic syndromes are already described. We did not determine any mutations past VE . Moreover, we sequenced other genes frequently mutated in melanoma, including, Nras , c kit , and Pten and didn’t acquire de novo mutations in these genes.
We also found that resistance to BRAF inhibitors was not connected to improvements in copy variety of Braf, Nras, c kit, or Pten . We noted that short term treatment with at mM led to a reduce in CRAF protein ranges in Lu cells, whereas CRAF ranges remained regular or in some cases even greater inside the resistant cells . Similarly, knockdown of BRAF employing shRNA, led to a rise in CRAF protein levels in the two the parental and resistant cells . We subsequent examined Raf Inhibitor selleck chemicals the likelihood that CRAF might be mediating ERK activation in response to BRAF inhibition . Lentiviral mediated infection of Lu R cells with CRAF shRNA inhibited CRAF expression, but had no effect on ERK activation . Remedy of CRAF shRNAinfected cells with had no result on phospho ERK ranges, indicating that resistant cells can activate the MAPK pathway independently of BRAF and CRAF. Similarly, infection of Lu R cells with three various ARAF shRNAs led to knockdown of this RAF isoform, but had no effect on phospho ERK .
Inhibition of BRAF activity by in conjunction with ARAF knockdown didn’t preclude phosphorylation of ERK in Lu R cells . Provided that resistant cells can activate ERK regardless of inhibition of either one particular or two RAF isoforms, we hypothesized that these cells only Semagacestat require one active RAF isoform to activate the MAPK pathway. To test this hypothesis, we sequentially contaminated Lu R cells with lentivirus carrying shRNAs against CRAF followed by infection with shRNAs towards ARAF . Simultaneous shRNA mediated inhibition of CRAF and ARAF did not have a significant impact on phospho ERK levels; however, therapy of those cells with mM resulted in downregulation of ERK phosphorylation .