4. Capítulo IV Propuesta
4.5.3. Plan de comunicación de estrategias de marketing educacional
The previous ODE models of the S. pombe mating-response pathway were limited when understanding the pathway as a whole, as only reactions describing the receptor R, Gα and RGS species were included. The transcriptional pathway is more complex than this and therefore, the latest version of the model by Croft et al. (2013) was extended to include all downstream signalling components upto and including Spk1 (a MAPK). As a result, a complete model termed the KR model was created, which was developed on experimental data collected both in this study and from the literature. The KR model could qualitatively reproduce the signalling characteristics of the end-point (assayed after 16 h of P-factor treatment) experimental data, which motivated its use as a predictive tool. When Gap1 was available in vitro Rgs1 acts as a negative and positive regulator of signalling [190]. The KR model predicted that by deletion of Gap1 (the negative regulator of Ras1), Rgs1 acts as a sole negative regulator. The equivalentin vitroexperiment showed that in cells treated with ≤100 nM P-factor, Rgs1 did in fact act as a negative regulator however, the increasing non-viability of cells prevented a typical sigmodal response at higher concentrations. Cell lysis was prevented by deletion of Scd1 (a GEF for CDC42), which transduces signalling via the MAP3K Byr2. In∆scd1∆gap1 strains cell lysis did not prevent sigmodial response to P-factor and indicated that Rgs1 acted as a sole negative regulator of signalling when treated with >100 nM, consistent with the KR model prediction.
The KR model was also simulated over time and compared with the equivalentin vitro time-series β-galactosidase activity experiments. The model displayed qualitative trends with the experimental data; showing that the number of response units increased with ligand concentration. However, the model was unable to capture the plateau in β-galactosidase
activity observed in vitro. The plateau in response suggested that lacZ, the gene that encodesβ-galactosidase, was no longer being transcribed. Attenuation of GPCR signalling occurs if the ligand (P-factor) is removed or the GPCR (Mam2) prevented from activating the G protein. Commonly, GPCRs are internalised following ligand-binding, to prevent continual stimulation and control the amount of activation through the pathway. The KR model was then simulated with a simple receptor desensitisation term that inactivated ligand-bound receptors, which showed a qualitative agreement with the time course data but lacked agreement with the end point data. The kinase(s) or processes involved in receptor desensitisation (including internalisation) are unknown and therefore, the identification of the protein(s) responsible for this process will be the focus of the next chapters.
The role of Cki1, Cki2 and Cki3 in
P-factor signalling
4.1
Background
The KR model developed in chapter 3 was unable to replicate the temporal plateau in β-galactosidase activity produced by WT (sxa2>lacZ) cells following treatment with P- factor (Figure 3.30). The plateau in response suggested that lacZ, the gene that encodes β-galactosidase, was no longer being transcribed. As stated in the Introduction, numerous mechanisms exist for terminating GPCR signalling, primarily via the removal of the ligand or inactivation of the receptor. M-typeS. pombe cells secrete Sxa2, a serine carboxypep-
tidase, in response to P-factor stimulation, which hydrolyses the extracellular pheromone [218, 267]. The sxa2− strain used in this time-course assay was unable to hydrolyse P- factor, yet still exhibited a transcriptional plateau, suggesting that other mechanisms were responsible for signal termination.
GPCRs are often internalised following phosphorylation of the intracellular receptor domains in response to ligand-binding to prevent further signal transduction via the G protein. Mam2, the M-type S. pombe pheromone receptor, is internalised in response to
P-factor [173]. However, the specific kinase(s) that promote this process have not yet been described. This chapter seeks to identity and characterise potential Mam2 kinase(s) in the M-typeS. pombe pheromone-response pathway.
YCK1 MSM---PIASTT--LAVNNLTN-ING---NANFNVQ-AN----KQLHHQAVDSPARSSMTA YCK2 MSQVQSPLTATNSGLAVNN--NTMNSQMPNRS-NVRLVNGTLPPSLH---V-S---SNLNH CKI1 MS---G---Q--- CKI2 MN---S---Q--- CKI3 MS---TTS---S---H--- YCK1 TTAANSN---SNS-SRDDSTIVGLHYKIGKKIGEGSFGVLFEGTNMINGVPVAIKFEPRKT YCK2 NTG-NSSASYSGSQSRDDSTIVGLHYKIGKKIGEGSFGVLFEGTNMINGLPVAIKFEPRKT CKI1 ---NN---VVGVHYKVGRRIGEGSFGVIFEGTNLLNNQQVAIKFEPRRS CKI2 ---TS---VVGVHYRVGRKIGEGSFGVIFDGMNLLNNQLIAIKFEPKKS CKI3 ---SN---VVGVHYRVGKKIGEGSFGMLFQGVNLINNQPIALKFESRKS YCK1 EAPQLRDEYKTYKILNGTPNIPYAYYFGQEGLHNILVIDLLGPSLEDLFDWCGRKFSVKTV
YCK2 EAPQLKDEYRTYKILAGTPGIPQEYYFGQEGLHNILVIDLLGPSLEDLFDWCGRRFSVKTV
CKI1 DAPQLRDEYRTYKLLAGCTGIPNVYYFGQEGLHNILVIDLLGPSLEDLLDLCGRKFSVK-T
CKI2 EAPQLRDEYRTYKLLVGNAGIPNVYYFGQEGLHNILVIDLLGPSLEDLFEWCGRRFSVK-T
CKI3 EVPQLRDEYLTYKLLMGLPGIPSVYYYGQEGMYNLLVMDLLGPSLEDLFDYCGRRFSPK-T
YCK1 VQVA-VQMITLIEDLHAHDLIYRDIKPDNFLIGRPGQPDANNIHLIDFGMAKQYRDPKTKQ YCK2 VQVA-VQMITLIEDLHAHDLIYRDIKPDNFLIGRPGQPDANKVHLIDFGMAKQYRDPKTKQ CKI1 VAMAAKQMLARVQSIHEKSLVYRDIKPDNFLIGRPNSKNANMIYVVDFGMVKFYRDPVTKQ CKI2 VAMTAKQMLSRVQTIHEKNLVYRDIKPDNFLIGRPSSRNANMVYMVDFGMAKYYRDPKTKQ CKI3 VAMIAKQMITRIQSVHERHFIYRDIKPDNFLIGFPGSKTENVIYAVDFGMAKQYRDPKT-- YCK1 HI--PYREKKSLSGTARYMSINTHLGREQSRRDDMEALGHVFFYFLRGHLPWQGLKAPNNK
YCK2 HI--PYREKKSLSGTARYMSINTHLGREQSRRDDMEAMGHVFFYFLRGQLPWQGLKAPNNK
CKI1 HI--PYREKKNLSGTARYMSINTHLGREQSRRDDLEALGHVFMYFLRGSLPWQGLKAATNK
CKI2 HI--PYSERKSLSGTARYMSINTHLGREQSRRDDLESLGHVFMYFLRGSLPWQGLKAANNK
CKI3 HVHRPYNEHKSLSGTARYMSINTHLGREQSRRDDLESMGHVFMYFLRGSLPWQGLKAATNK
YCK1 QKYEKIGEKKRSTNVYDLAQGLPVQFGRYLEIVRSLSFEECPDYEGY-RKLLLSVL-DDLG YCK2 QKYEKIGEKKRLTNVYDLAQGLPIQFGRYLEIVRNLSFEETPDYEGY-RMLLLSVL-DDLG CKI1 QKYERIGEKKQSTPLRELCAGFPEEFYKYMHYARNLAFDATPDYD-YLQGLFSKVLER-LN CKI2 HKYEKISEKKQSTSISELCAGFPNEFSKYMTYVRSLEFDEEPDYA-FLQELFDDVL-RANG CKI3 QKYEKIGEKKQVTPLKELCEGYPKEFLQYMIYARNLGYEEAPDYD-YLRSLFDSLLLR-IN
YCK1 ETADGQYDWMKLNDGRGWDLN---INKKPN---LHG---Y-GHPN---PPNE YCK2 ETADGQYDWMKLNGGRGWDLS---INKKPN---LHG---Y-GHPN---PPNE CKI1 TTEDENFDWNLLNNGKGWQ-SL-K---SR-N--A-ET-EN---QRS-SK-PP-A CKI2 DTNDGVYDWMLLNDGKGWE-SS-S---SHFSVVAMKRRKNYLGL-NVVQNDDSR--- CKI3 ETDDGKYDWTLLNNGKGWQYSAAKQHVVQRRHT----QG-TN---N--RRQ-STIPPYA YCK1 -KSR-KH--RNKQLQM---QQL--QMQ-Q--L--Q---Q-Q-Q-Q---Q YCK2 -KSK-RH--RSKNHQYSSPDHHHHYNQQQ--QQQ-Q--A--QA----QAQAQAQAK---VQ
CKI1 PKLESK----SPALQ---NHA-STQNVVSK-RSDYE---KPFAEPHLN-S--AS-DSAE
CKI2 -K---KN---S-TLQ---TQNM--RFKSSYGV--R--G-PR-NYS---SFD-AL
CKI3 -RTR-QNLLSSPSKQ--TPVNNV-VDASVATQ-K-D-GIPGKA-ASPQVQQQQQTS--SAQ YCK1 QQYAQK-T---E-ADMR-NSQ--YK--P---KLDPTS---YEAYQH-- YCK2 QQQLQQ-A---Q-AQQQAN-R--YQLQPDDSHYDEERE--ASKLDPTS---YEAYQQ-- CKI2 ---PSKNA-PLVRQ--EQSASKKTIYA-HSSR-GYDRVRPMYVSQ--PSNNA-VGV-NHPN CKI3 QQQPQR---V----EQPA---P-Q---TTQ--PTQ---VD--- YCK1 ---Q---T--Q-Q---K--YL--QEQ-QKRQQQQKLQEQQLQEQQLQQQQQQQQQLRA-TG YCK2 ---Q---T--Q-Q---K--YA--QQQ-QK-QMQQK--SKQF---ANTG CKI1 DSSEERVTREQVQNATKETEAP-K-K-KK---S---F--- CKI2 D---NS--DSEA---KG---G---F--- CKI3 ---T--Q-Q-AAKP--APSKEKSRK---K---F--- YCK1 QPPSQPQAQTQSQQFGARYQPQQ-QPSAALRTPEQHP-N--DD-NSSLAASHKGFFQKLGC YCK2 ---AN--GQTN---KY-PYNAQPTA---NDEQNAKNAAQDRNSN-KSS-KGFFSKLGC CKI1 ---W---AS---I---LSC CKI2 ---F---D---M---ICC CKI3 ---H---LR---L---LSC YCK1 -C---* YCK2 -C---* CKI1 -C-SGSNEDT* CKI2 RCFS---* CKI3 RCFS---*
CKI1 ---PNQNSLP--NPPTETKATT-TV----PDRSG---LATNQ--P---APVDV--H--
1 61 62 122 123 183 184 244 245 305 306 366 367 427 428 488 489 549 550 610 611 671 672 683
Figure 4.1: 67-69% homology between the S. cerevisiae proteins YCK1 and YCK2, with the S. pombe proteins Cki1, Cki2 and Cki3. Sequences were aligned using MutiAlin version 5.4.1 [268]. Gaps (-) were included to optimise the alignment, identical residues are highlighted in red, conservative changes in blue and non-conservative