T cells stimulated by antigen receptor, growth factor cytokines such as IL-2 or chemotactic chemokines such as S D F la all trigger an independent array of signalling molecules that culminate in the activation of a number of common S/T kinases. As discussed in chapter 1 SAT kinases make up 2% of the coded genes within the genome and are responsible for over 90% of the protein phosphorylation events within a typical cell. This huge degree of protein phosphorylation has paradoxically made investigation of targets of S/T kinase activity difficult, as significant stimulus dependent phosphorylation events are hidden in a background of stimulus independent ‘house-keeping’ S/T residue phosphorylation.
Anti-phospho-tyrosine antisera were key tools in the elucidation of the complex networks of lymphocyte specific adapter proteins that couple antigen receptors to downstream signalling molecules.(Chan et al., 1991; Jackman et al., 1995; Zhang et al., 1998a). Our aim was to be able to devise anti sera that could be used to identify phosphorylated protein substrates of S/T kinases in T cells. Using this anti serum we hoped to increase our understanding of the signalling pathways that govern T cell activation in response to antigenic stimulation. We felt it was necessary to use biochemical analysis in intact cells to
define S/T kinase networks in T cells rather than in vitro analysis in cell extracts. The
reason for this preference is that the substrate specificity of a kinase may be mediated by the larger structure of the protein target than simply the phosphorylation pocket shape. There may be other regions of the target protein that mediate binding of the Serine kinase to
its target. Finally, localisation of both the target protein and potential kinase may determine
whether they will be able to interact under in vivo physiological conditions. The cellular
distribution of PKB is tightly controlled within a lymphocyte with an initial protein localisation at the time of activation followed by rapid transit to the nucleus (Astoul et al., 1999). Similarly PKC family members can be chaperoned to particular cellular regions by interacting proteins including RACKs (receptors for activated C kinase) and STICKs (substrates that interact with C kinase) (Bauman and Scott, 2002).
In chapter 3 we describe making an antiserum termed PAP-1 which was obtained from rabbits immunised with a phospho-peptide RARTSpSFAEP that corresponds to phosphorylation site of Serine 21 in GSK3a. We hoped that in addition to identifying phosphorylated GSK3a this antibody would also identify a subset of proteins which are substrates for S/T kinases activated in T cells in response to antigen receptor ligation. The results show that PAP-1 can be used to define other substrates for antigen receptor regulated kinases that are phosphorylated in parallel to G SK 3a and which contain a sequence structurally similar to GSK3a Ser21. PAP-1 thus recognises a tier of serine kinase substrates regulated by antigen receptors.
The immunising peptide for PAP-1 (GSK3a Ser21) is a PKB substrate but it should be emphasised that PAP-1 is also able to recognise proteins phosphorylated via PKC pathways in T lymphocytes. This prevents PAP-1 for being used as a reporter for activation of a particular kinase pathway. Moreover, in the context of PKB, PAP-1 only recognises a subset of PKB substrates. Oligopeptide competition experiments demonstrate the high
degree of specificity PAP-1 has for the flanking residues around the target serine. This restricts the specificity of PAP-1 but the optimal oligopeptide target sequences for many AGC kinases are very similar particularly between the PKB, p90-RSK and MSK families that show a high degree of homology in their respective kinase domains. However, this lack of specificity for a particular serine kinase pathway does not preclude PAP-1 from being a useful tool to identify novel S/T kinase substrates in TcR activated cells. Experiments with PAP-1 allowed us to identify two novel substrates for TcR regulated S/T kinases in human T lymphocytes. This work has led us to conclude that PAP-1 is a useful tool for exploring novel S/T kinase substrates in lymphocytes. It could be useful to use PAP-1 and similar antisera to compare the phospho-proteome of primary lymphocytes of different lineages
(i.e. CD4+ and CD8+ T cells) or of different stages of development/differentiation (i.e.:
naïve, effector and memory T and B cells).
In chapter 4 we discussed the strategy for purification of two of the PAP-1 identified proteins, PAPlp55 and PAPlp85. We were unable to immunoprecipitate proteins using the PAP-1 antiserum and elected to use column chromatography to partially purify both proteins before using two-dimensional gel electrophoresis as a final step. Protein spots were trypsinised and identified by mass spectroscopy. PAPlp55 was identified as the Sterile alpha motif - Src homology 3 (SAM-SH3) containing protein SLY, PAPlp85 was identified as the cytokine precursor protein proIL-16. Analysing the amino acid sequences identified two likely PAP-1 identification sites. Using the Scansite program, Ser27 in SLY conforms to a site of likely PKC phosphorylation and S eri44 in proIL-16 conforms to a site of likely PKB phosphorylation, both consistent with the findings of experiments in chapter
3. Despite being candidate sites for different protein kinases as defined by Scansite, there are a number of similarities between the Ser27 site in SLY and the Serl44 site of proIL-16 respectively; both contain two serines followed by a phenylalanine residue. The similarity was confirmed by the peptide competition experiment in figure 4.7; phospho-oligopeptides from SLY and proIL-16 were both able to compete out the PAPlp55 and p85 bands. SLY is a member of a family of SAM-SH3 containing proteins: examining their sequences in a number of species shows a homologous site to the Ser27 position in a number of SAM-SH3 family proteins that is preserved across species (Fig 6.1). proIL-16 has one close family member, neuronal-proIL-16 that is highly homologous but contains an additional N- terminal PDZ domain, the S erl44 site is preserved between these two proteins. The sequences of proIL-16 have been published for the major primates (Bannert et al., 1998). Like Ser27 in SLY, the Serl44 site is conserved, although in some animals the arginine at the -5 position is replaced with a similarly positively charged lysine.
Work by Manning et al (Manning et al., 2002) has suggested it is possible to identify S/T kinase substrates using a bioinformatic method. We used the known properties of PAP-1 and the information gleaned from 2D-SDS PAGE to see how useful a bioinformatic approach would have been in identifying both proIL-16 and SLY.
If we interrogate the Swissprot database for proteins containing a peptide sequence
corresponding to that of Ser21 in GSK3a, then a short list of 13 proteins with a predicted molecular weight of 85kDa was obtained. Similarly nine candidates for a 55kDa protein were obtained. The p85 list contained proIL-16 but the p55 list did not contain SLY
because this sequence is not yet in the database. The p55 list did contain a close relative of SLY: SAMSN-1 that contains a site homologous to Ser27 of SLY (Fig 6.1). SAMSN-1 transfected into antigen receptor stimulated A20 lymphocytes is identified by PAP-1 on Western blot (E.Astoul, personal communication). This highlights the advantages and disadvantages of such an approach. To obtain cDNA sequences suitable for cellular transfection per candidate is not trivial especially when there is a chance that the actual protein is not on the list either because it is not listed within the scanned protein database or because the search criteria are too strict. However, data base mining can identify new candidates such as SAMSN-1 that were not identified by our protein purification approach.
In summary, the present data show that PAP-1 has been invaluable for T cell studies. Similarly, the BPS antiserum produced by Cell Signalling Technologies has allowed a number of laboratories to identify novel substrates for PKB (Basu et al., 2003; Berwick et al., 2002; Kane et al., 2002; Manning et al., 2002). There are merits and disadvantages of using ‘wet laboratory’ techniques versus bioinformatic approaches to identify candidate proteins indicated by the antisera. Advances in technology will benefit both techniques as protein databases become more comprehensive and protein purification techniques become more sophisticated with mass spectrometry able to identify protein derived from smaller samples.
FIG 6.1
PAP-1 recognition site s ly s ly mouse sly_fugu s ly gallu s ssnl_human ssn l mouse 40 * 60
P’A'jEK EFX >::D N IPED D SG V PT?|DA G | PVVvE K E F '± 0 N IP E D D S G V L T P |j3g| P -V ^E K E It'ÏSENV--- AG— A P|DPI PVT^EKEFX^ZSNIPSDEPSSAGP^SAAgSG LSKPDDSTEAHEGDP— T N - G S G E Q s R s X ;s f| d d s i e| id r e l— t n- g s. ^q sBs s s ly sly_mouse sly_fugu s l y j a l l u s ssnl_human ssn l mouse » 120 ïz m a d t l e S g s a s p t s p d y s l d s p g p ; 140 * 1 6 0 s e q e e h e l p v lBr q a^ s e l cSp s p g s g- ^g e e p h 'E Q E E R E P PS L gR gT ^SE L C gPG PG S G -gE L E E SP A g GMGDTLEi^SASPTSPDCSLDSPGP: ^R--- G K Q A E V E jjE G C P S P L P S A G T E E P S H g p S sW E M E E C A H P S v S R Q L ^S E G L B P G P T S Q Y lP R L E D G S i fK D E E D G '|N A H P Y R N S D P V IG -T H Æ sg K A S D S M D S L Y S G < 2 i-S ^ IT S C |D G T S N R : "k e e e s g^e a l p y r n s d?m ig-t h t3 Rs2k a s d s m d s l y s g< 2 | -s" i t s c§d g t s n r! •HitHLKL 1 9 6 1 9 6 93 1 9 9 1 8 6 1 8 6 sly sly_mouse sly_fugu s l y j a l l u s ssnl_human ssn l mouse 2 2 0 * 2 4 0 * 2 6 0 q k g d v iq iie k p p v g t o l g l l n g k v g s f k f i y v d v l p e e a v g h a r p s r r q s k g k r p k p kJ h ï lJ q k g d v iq iv e k p p v g t w l g l l n g k l g s f k f iy v d v l p e e a v g p v r p s r r q s k g k r p k p kÎ Sh^li xxmxxmxxx»QQ20QQæ0QQ0ü{mxxmxxxmmmxxmxRLKP: :<KGDIIDIICKTPMGPMT(3€;NNKVGNFKFIYVDVISEEEAAPKKIKANRRSKSK-KS KKGDIIDIICKTPMGPWTGMLNNKVGNFKFIYVDVISEEEAAPKKIKVPRSRRRE-NHQI 2 9 9 2 8 5 2 8 5 s ly sly_mouse sly_fugu s l y j a l l u s ssnl_human ssn l mouse 320 * 340 jLDTDYDTGSEEAE|GAESSQEPVAHTVSEPKvTl *Li| dYDTGSEEAE£GAESSQEPVAHTVSEPKV*i" EGaE— E G p — SGjjDEAjJlAGTEEQLQGilSiJSGAP- 380 380
IllSdY D T S S - EAE?GDGTAEQ— HS PSE P K g S i B N FffEE EIIQ EQ EN |PEPLSLSSD ISLN K SQ LD Îc ïS L g D E E W E H E K iS V P L S S N P D I-L S A S Q L E iC
^l p s p a g q p q; ^s2a e— : 3 80 IS§GNSDNG!àEDLl>SENLSEMVHK0I0TEPSD : 373 IS^NSDNGtSEDLgSENLSDMVQKgAHTESSD : 372
Fig 6.1: The PAP-1 recognition site is conserved in SLY and SAMSN-1 from humans to fish.
The am ino acid sequence o f SLY and SAM SN-1 (SSN -1) w as com pared form hum ans, m ice and fish. A ll proteins analysed show ed a high degree o f hom ology at the PAP-1 recognition site
BIBLIOGRAPHY
Abtahian, F., Guerriero, A., Sebzda, E., Lu, M.M., Zhou, R., Mocsai, A., Myers, E.E., Huang, B., Jackson, D.G., Ferrari, V.A., Tybulewicz, V., Lowell, C.A., Lepore, J.J., Koretzky, G. A. and Kahn, M.L. (2003) Regulation of blood and lymphatic vascular
separation by signaling proteins SLP-76 and Syk. Science, 299, 247-251.
Acuto, O. and Cantrell, D. (2000) T cell activation and the cytoskeleton. Annu Rev
Immunol, 18, 165-184.
Alarcon, B., Gil, D., Delgado, P. and Schamel, W.W. (2003) Initiation of TCR signaling:
regulation within CD3 dimers. Immunol Rev, 191, 38-46.
Alessi, D.R., Deak, M., Casamayor, A., Caudwell, F.B., Morrice, N., Norman, D.G., Gaffney, P., Reese, C.B., MacDougall, C.N., Harbison, D., Ashworth, A. and Bownes, M. (1997a) 3-Phosphoinositide-dependent protein kinase-1 (PDKl):
structural and functional homology with the Drosophila DSTPK61 kinase. Curr
Biol, 7, 776-789.
Alessi, D R., James, S.R., Downes, C.P., Holmes, A.B., Gaffney, P.R., Reese, C.B. and Cohen, P. (1997b) Characterization of a 3-phosphoinositide-dependent protein
kinase which phosphorylates and activates protein kinase Balpha. Curr Biol, 7, 261-
269.
Alessi, D.R., Saito, Y., Campbell, D.G., Cohen, P., Sithanandam, G., Rapp, U., Ashworth, A., Marshall, C.J. and Cowley, S. (1994) Identification of the sites in MAP kinase
kinase-1 phosphorylated by p74raf-l. Embo J, 13, 1610-1619.
Altman, A. and Villalba, M. (2003) Protein kinase C-theta (PKCtheta): it's all about
location, location, location. Immunol Rev, 192, 53-63.
Anderson, G. and Jenkinson, E.J. (2001) Lymphostromal interactions in thymic
development and function. Nat Rev Immunol, 1, 31-40.
Astoul, E., Laurence, A.D., Totty, N., Beer, S., Alexander, D.R. and Cantrell, D.A. (2003) Approaches to define antigen receptor-induced serine kinase signal transduction
pathways. J Biol Chem, 278, 9267-9275.
Baeuerle, P.A. and Baltimore, D. (1996) NF-kappa B: ten years after. Cell, 87, 13-20. Baier, M., Bannert, N., Werner, A., Lang, K. and Kurth, R. (1997) Molecular cloning,
sequence, expression, and processing of the interleukin 16 precursor. Proc Natl
A cadSci U S A , 94, 5273-5277.
Baier, M., Werner, A., Bannert, N., Metzner, K. and Kurth, R. (1995) HIV suppression by
interleukin-16. Nature, 378, 563.
Balendran, A., Casamayor, A., Deak, M., Paterson, A., Gaffney, P., Currie, R., Downes, C.P. and Alessi, D.R. (1999) PDKl acquires PDK2 activity in the presence of a
synthetic peptide derived from the carboxyl terminus of PRK2. Curr Biol, 9, 393-
404.
Bannert, N., Adler, H.S., Werner, A., Baier, M. and Kurth, R. (1998) Molecular cloning and sequence analysis of interleukin 16 from nonhuman primates and from the
mouse. Immunogenetics, 47, 390-397.
Bassing, C.H., Swat, W. and Alt, F.W. (2002) The mechanism and regulation of
chromosomal V(D)J recombination. Cell, 109 Suppl, S45-55.
Basson, M.A. and Zamoyska, R. (2000) The CD4/CD8 lineage decision: integration of
signalling pathways. Immunol Today, 21, 509-514.
Basu, S., Totty, N.F., Irwin, M.S., Sudol, M. and Downward, J. (2003) Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation
of p73-mediated apoptosis. Mol Cell, 11, 11-23.
Bauman, A.L. and Scott, J.D. (2002) Kinase- and phosphatase-anchoring proteins:
harnessing the dynamic duo. Nat Cell Biol, 4, E203-206.
Beer, S., Simins, A.B., Schuster, A. and Holzmann, B. (2001) Molecular cloning and characterization of a novel SH3 protein (SLY) preferentially expressed in lymphoid
cells. Biochim Biophys Acta, 1520, 89-93.
Bertolotto, C., Maulon, L., Filippa, N., Baier, G. and Auberger, P. (2000) Protein kinase C theta and epsilon promote T-cell survival by a rsk-dependent phosphorylation and
inactivation of BAD. J Biol Chem, 275, 37246-37250.
Berwick, D.C., Hers, 1., Heesom, K.J., Moule, S.K. and Tavare, J.M. (2002) The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary
Biggs, W.H., 3rd, Meisenhelder, J., Hunter, T., Cavenee, W.K. and Arden, K.C. (1999) Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the
winged helix transcription factor FKHRl. Proc Natl Acad Sci U S A , 96, 7421-
7426.
Boniface, J.J., Reich, Z., Lyons, D.S. and Davis, M.M. (1999) Thermodynamics of T cell receptor binding to peptide-MHC: evidence for a general mechanism of molecular
scanning. Proc Natl Acad Sci U S A , 96, 11446-11451.
Bossi, G., Trambas, C., Booth, S., Clark, R., Stinchcombe, J. and Griffiths, G.M. (2002)
The secretory synapse: the secrets of a serial killer. Immunol Rev, 189, 152-160.
Bouillet, P., Purton, J.F., Godfrey, D.I., Zhang, L.C., Coultas, L., Puthalakath, H., Pellegrini, M., Cory, S., Adams, J.M. and Strasser, A. (2002) BH3-only Bcl-2
family member Bim is required for apoptosis of autoreactive thymocytes. Nature,
415, 922-926.
Brennan, P., Babbage, J.W., Burgering, B.M., Groner, B., Reif, K. and Cantrell, D.A. (1997) Phosphatidylinositol 3-kinase couples the interleukin-2 receptor to the cell
cycle regulator E2F. Immunity, 7, 679-689.
Brennan, P., Babbage, J.W., Thomas, G. and Cantrell, D. (1999) p70(s6k) integrates phosphatidylinositol 3-kinase and rapamycin-regulated signals for E2F regulation in
T lymphocytes. Mol Cell Biol, 19,4729-4738.
Bromley, S.K., Burack, W.R., Johnson, K.G., Somersalo, K., Sims, T.N., Sumen, C., Davis, M.M., Shaw, A.S., Allen, P.M. and Dustin, M.L. (2001) The immunological
synapse. Annu Rev Immunol, 19, 375-396.
Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S., Anderson, M.J., Arden, K.C., Blenis, J. and Greenberg, M.E. (1999) Akt promotes cell survival by
phosphorylating and inhibiting a Forkhead transcription factor. Cell, 96, 857-868.
Bryant, P.W., Lennon-Dumenil, A.M., Fiebiger, E., Lagaudriere-Gesbert, C. and Ploegh,
H.L. (2002) Proteolysis and antigen presentation by MHC class II molecules. Adv
Immunol, 80, 71-114.
Bunnell, S.C., Diehn, M., Yaffe, M.B., Findell, P R., Cantley, L.C. and Berg, L.J. (2000) Biochemical interactions integrating Itk with the T cell receptor-initiated signaling
Burgering, B.M. and Kops, G J. (2002) Cell cycle and death control: long live Forkheads.
Trends Biochem Sci, 27, 352-360.
Byth, K.F., Conroy, L.A., Howlett, S., Smith, A.J., May, J., Alexander, D.R. and Holmes, N. (1996) CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B
cell maturation. J Exp Med, 183, 1707-1718.
Cantley, L.C. and Neel, B.C. (1999) New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT