Phosphorylation of Ser2078 Modulates the Notch2 Function in 32D Cell Differentiation
2001; Elsevier BV; Volume: 276; Issue: 48 Linguagem: Inglês
10.1074/jbc.m104703200
ISSN1083-351X
AutoresJulia Inglés‐Esteve, Lluı́s Espinosa, Laurie A. Milner, Carmé Caelles, Anna Bigas,
Tópico(s)TGF-β signaling in diseases
ResumoNotch signaling is involved in the regulation of many cell fate determination events in both embryonic development and adult tissue homeostasis. We previously demonstrated that Notch1 and Notch2 molecules inhibit myeloid differentiation in a cytokine-specific manner and that the Notch cytokine response domain is necessary for this functional specificity. We have now investigated the putative role of phosphorylation in the activity of Notch in response to cytokine signals. Our results show that the granulocyte colony-stimulating factor (G-CSF) stimulation of 32D cells expressing the intracellular Notch2 protein induces phosphorylation at specific sites of this molecule, rendering the molecule inactive and permitting differentiation of these cells. In contrast, when cells are stimulated with granulocyte macrophage colony-stimulating factor (GM-CSF), intracellular notch2 is not phosphorylated at these residues and differentiation is inhibited. We also show that deletion of the Ser/Thr-rich region between amino acids 2067 and 2099 abrogates G-CSF-induced phosphorylation and results in a molecule that inhibits differentiation in response to either G-CSF or GM-CSF. Our results further indicate that Ser2078 is a critical residue for phosphorylation and modulation of Notch2 activity in the context of G-CSF-induced differentiation of 32D cells. Notch signaling is involved in the regulation of many cell fate determination events in both embryonic development and adult tissue homeostasis. We previously demonstrated that Notch1 and Notch2 molecules inhibit myeloid differentiation in a cytokine-specific manner and that the Notch cytokine response domain is necessary for this functional specificity. We have now investigated the putative role of phosphorylation in the activity of Notch in response to cytokine signals. Our results show that the granulocyte colony-stimulating factor (G-CSF) stimulation of 32D cells expressing the intracellular Notch2 protein induces phosphorylation at specific sites of this molecule, rendering the molecule inactive and permitting differentiation of these cells. In contrast, when cells are stimulated with granulocyte macrophage colony-stimulating factor (GM-CSF), intracellular notch2 is not phosphorylated at these residues and differentiation is inhibited. We also show that deletion of the Ser/Thr-rich region between amino acids 2067 and 2099 abrogates G-CSF-induced phosphorylation and results in a molecule that inhibits differentiation in response to either G-CSF or GM-CSF. Our results further indicate that Ser2078 is a critical residue for phosphorylation and modulation of Notch2 activity in the context of G-CSF-induced differentiation of 32D cells. intracellular Notch intracellular Notch1 intracellular Notch2 Notch cytokine response nuclear localization signal serine threonine-rich granulocyte colony-stimulating factor granulocyte macrophage colony-stimulating factor polymerase chain reaction SDS-polyacrylamide gel electrophoresis mitogen-activated protein kinase Notch molecules are highly conserved transmembrane proteins that, through their participation in cell-cell interactions, play critical roles in cell fate decisions during many developmental processes (reviewed in Ref. 1Egan S.E. St Pierre B. Leow C.C. Curr. Top. Microbiol. Immunol. 1998; 228: 273-324Crossref PubMed Google Scholar). Four distinct Notch homologs have been identified in mammals (2Ellisen L.W. Bird J. West D.C. Soreng A.L. Reynolds T.C. Smith S.D. Sklar J. Cell. 1991; 66: 649-661Abstract Full Text PDF PubMed Scopus (1438) Google Scholar, 3Weinmaster G. Roberts V.J. Lemke G. Development. 1992; 116: 931-941Crossref PubMed Google Scholar, 4Lardelli M. Dahlstrand J. Lendahl U. Mech. Dev. 1994; 46: 123-136Crossref PubMed Scopus (260) Google Scholar, 5Uyttendaele H. Marazzi G. Wu G. Yan Q. Sassoon D. Kitajewski J. Development. 1996; 122: 2251-2259Crossref PubMed Google Scholar) and the extent to which they have specific functions is a theme of current investigation. The Notch1 and 2 molecules are synthesized as single precursor proteins that are cleaved in the Golgi to become functional heterodimeric receptors present on the cell surface. Notch ligands are expressed on the surface of neighboring cells and also on the same cell as the receptor, with the relative dosage of receptor and ligand being an important determinant of Notch activity (6Heitzler P. Simpson P. Cell. 1991; 64: 1083-1092Abstract Full Text PDF PubMed Scopus (581) Google Scholar). The productive interaction of Notch receptor with ligand results in further proteolytic cleavage, with release and nuclear translocation of the intracellular domain of Notch (Notch-IC),1 which functions as a transcriptional regulator (reviewed in Ref. 7Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Crossref PubMed Scopus (224) Google Scholar). Truncated Notch molecules corresponding to Notch-IC (i.e. lacking the transmembrane and extracellular domains) behave as constitutively active molecules, and thus have been used extensively to study Notch function in many vertebrate and invertebrate systems (8Fortini M.E. Rebay I. Caron L.A. Artavanis-Tsakonas S. Nature. 1993; 365: 555-557Crossref PubMed Scopus (258) Google Scholar, 9Struhl G. Fitzgerald K. Greenwald I. Cell. 1993; 74: 331-345Abstract Full Text PDF PubMed Scopus (409) Google Scholar, 10Kopan R. Nye J.S. Weintraub H. Development. 1994; 120: 2385-2396Crossref PubMed Google Scholar, 11Milner L.A. Bigas A. Kopan R. Brashem-Stein C. Bernstein I.D. Martin D.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13014-13019Crossref PubMed Scopus (170) Google Scholar). Notch activity generally, but not exclusively, leads to the inhibition of a differentiating signal, thereby preserving progenitors that are capable of responding to subsequent signals and generating cell diversity.Despite the extraordinary conservation of Notch structural domains, several observations support the lack of complete functional redundancy. Inactivation of different Notch genes in mice has been very instructive in this regard; for instance,Notch1 and Notch2 null mice are embryonic lethals before day 11.5 (12Swiatek P.J. Lindsell C.E. del Amo F.F. Weinmaster G. Gridley T. Genes Dev. 1994; 8: 707-719Crossref PubMed Scopus (604) Google Scholar, 13Hamada Y. Kadokawa Y. Okabe M. Ikawa M. Coleman J.R. Tsujimoto Y. Development. 1999; 126: 3415-3424Crossref PubMed Google Scholar), and they show distinct phenotypic defects indicating that they cannot compensate for the inactivated molecule (14Lindsell C.E. Boulter J. diSibio G. Gossler A. Weinmaster G. Mol. Cell Neurosci. 1996; 8: 14-27Crossref PubMed Scopus (307) Google Scholar, 15Bao Z.Z. Cepko C.L. J Neurosci. 1997; 4: 1425-1434Crossref Google Scholar, 16Mitsiadis T.A. Lardelli M. Lendahl U. Thesleff I. J. Cell Biol. 1995; 130: 407-418Crossref PubMed Scopus (152) Google Scholar). Although the biochemical mechanisms remain elusive (17Kato H. Sakai T. Tamura K. Minoguchi S. Shirayoshi Y. Hamada Y. Tsujimoto Y. Honjo T. FEBS Lett. 1996; 2–3: 221-224Crossref Scopus (89) Google Scholar, 18Ordentlich P. Lin A. Shen C.P. Blaumueller C. Matsuno K. Artavanis Tsakonas S. Kadesch T. Mol. Cell. Biol. 1998; 18: 2230-2239Crossref PubMed Scopus (230) Google Scholar), these studies provide evidence for the specificity of Notch homolog function during mammalian development.Notch1, Notch2, and Notch3, as well as multiple DSL ligands, are expressed in hematopoietic cells (19Milner L.A. Kopan R. Martin D.I. Bernstein I.D. Blood. 1994; 83: 2057-2062Crossref PubMed Google Scholar, 20Singh N. Phillips R.A. Iscove N.N. Egan S.E. Exp. Hematol. 2000; 28: 527-534Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 21Tsai S. Fero J. Bartelmez S. Blood. 2000; 96: 950-957Crossref PubMed Google Scholar). Notch signaling has been shown to influence the development of lymphoid (22Robey E. Chang D. Itano A. Cado D. Alexander H. Lans D. Weinmaster G. Salmon P. Cell. 1996; 87: 483-492Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 23Pui J.C. Allman D. Xu L. DeRocco S. Karnell F.G. Bakkour S. Lee J.Y. Kadesch T. Hardy R.R. Aster J.C. Pear W.S. Immunity. 1999; 11: 299-308Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar, 24Radtke F. Ferrero I. Wilson A. Lees R. Aguet M. MacDonald H.R. J. Exp. Med. 2000; 191: 1085-1094Crossref PubMed Scopus (127) Google Scholar), myeloid (11Milner L.A. Bigas A. Kopan R. Brashem-Stein C. Bernstein I.D. Martin D.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13014-13019Crossref PubMed Scopus (170) Google Scholar,25Bigas A. Martin D.I. Milner L.A. Mol. Cell. Biol. 1998; 18: 2324-2333Crossref PubMed Scopus (183) Google Scholar, 26Carlesso N. Aster J.C. Sklar J. Scadden D.T. Blood. 1999; 93: 838-848Crossref PubMed Google Scholar, 27Schroeder T. Just U. EMBO J. 2000; 19: 2558-2568Crossref PubMed Scopus (114) Google Scholar), and erythroid cells (28Lam L.T. Ronchini C. Norton J. Capobianco A.J. Bresnick E.H. J. Biol. Chem. 2000; 275: 19676-19684Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29Shelly L.L. Fuchs C. Miele L. J. Cell. Biochem. 1999; 73: 164-175Crossref PubMed Scopus (101) Google Scholar) and to participate in the maintenance of the hematopoietic progenitor pool (30Jones P. May G. Healy L. Brown J. Hoyne G. Delassus S. Enver T. Blood. 1998; 92: 1505-1511Crossref PubMed Google Scholar, 31Varnum-Finney B. Purton L.E. Yu M. Brashem-Stein C. Flowers D. Staats S. Moore K.A. Le Roux I. Mann R. Gray G. Artavanis-Tsakonas S. Bernstein I.D. Blood. 1998; 91: 4084-4091Crossref PubMed Google Scholar, 32Han W. Ye Q. Moore M.A. Blood. 2000; 95: 1616-1625Crossref PubMed Google Scholar, 33Walker L. Lynch M. Silverman S. Fraser J. Boulter J. Weinmaster G. Gasson J.C. Stem Cells. 1999; 17: 162-171Crossref PubMed Scopus (102) Google Scholar). Despite the emerging evidence implicating Notch as a key mediator in hematopoiesis, the precise role of the different Notch homologs in regulating specific hematopoietic cell fate decisions remains unclear. Within the lymphoid system, Notch1 and Notch2 are preferentially expressed in thymus and spleen, respectively (3Weinmaster G. Roberts V.J. Lemke G. Development. 1992; 116: 931-941Crossref PubMed Google Scholar). However, T and B lymphocytes express both Notch1 and2, and in other hematopoietic cell types Notch1, 2, and 3 are frequently co-expressed, 2L. A. Milner, unpublished observations.2L. A. Milner, unpublished observations. suggesting that more than one Notch molecule may function simultaneously in a given cell.Recently it has been shown that Notch molecules are post-translationally modified by glycosylation (34Moloney D.J. Shair L.H. Lu F.M. Xia J. Locke R. Matta K.L. Haltiwanger R.S. J. Biol. Chem. 2000; 275: 9604-9611Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar) and phosphorylation (35Kidd S. Lieber T. Young M.W. Genes Dev. 1998; 12: 3728-3740Crossref PubMed Scopus (192) Google Scholar), adding further complexity to the regulation of Notch signaling. Glycosylation of Notch EGF repeats by fringe molecules is important in modulating Notch-ligand interactions (36Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (590) Google Scholar, 37Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (716) Google Scholar) and it is likely to contribute to ligand binding specificity of the different Notch homologs (38Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (335) Google Scholar).Phosphorylation is a widely used mechanism for regulating activity through signal transduction pathways. Recent reports have shown that a nuclear form of N1IC1 (Notch1-IC) is phosphorylated (39Redmond L. Oh S.R. Hicks C. Weinmaster G. Ghosh A. Nat. Neurosci. 2000; 3: 30-40Crossref PubMed Scopus (278) Google Scholar) and that ligand binding to Notch2 induces hyperphosphorylation and nuclear translocation of this molecule (40Shimizu K. Chiba S. Hosoya N. Kumano K. Saito T. Kurokawa M. Kanda Y. Hamada Y. Hirai H. Mol. Cell. Biol. 2000; 20: 6913-6922Crossref PubMed Scopus (141) Google Scholar). Furthermore, both phosphorylation and nuclear translocation appear to be necessary for oncogenic transformation mediated by Notch (41Ronchini C. Capobianco A.J. Oncogene. 2000; 19: 3914-3924Crossref PubMed Scopus (39) Google Scholar). Together, these findings suggest that phosphorylation may be an important factor in modulating intracellular signal transduction through the Notch pathway.Previously, we have reported that either expression of truncated Notch1 (N1ICΔOP) or ligand activation of the full-length Notch1 receptor inhibits G-CSF-induced differentiation of 32D myeloid progenitors (11Milner L.A. Bigas A. Kopan R. Brashem-Stein C. Bernstein I.D. Martin D.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13014-13019Crossref PubMed Scopus (170) Google Scholar, 42Li L. Milner L.A. Deng Y. Iwata M. Banta A. Graf L. Marcovina S. Friedman C. Trask B.J. Hood L. Torok Storb B. Immunity. 1998; 8: 43-55Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). However, N1ICΔOP does not inhibit differentiation induced by GM-CSF; and conversely, truncated Notch2 (N2ICΔOP) does not affect G-CSF-induced differentiation, but inhibits differentiation in response to GM-CSF (42Li L. Milner L.A. Deng Y. Iwata M. Banta A. Graf L. Marcovina S. Friedman C. Trask B.J. Hood L. Torok Storb B. Immunity. 1998; 8: 43-55Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). These latter studies also revealed that a specific region of the Notch molecule, designated the NCR (Notch cytokine response) region, confers this cytokine specificity. In the present study, we have investigated whether phosphorylation plays a role in regulating activity of the N2ICΔOP molecule. We find that N2ICΔOP is differentially phosphorylated in 32D cells exposed to G-CSF or GM-CSF. In the context of G-CSF stimulation, phosphorylation of N2ICΔOP correlates with its inability to inhibit differentiation. At least one of the N2ICΔOP residues phosphorylated in G-CSF-stimulated cells mapped to a serine/threonine-rich (STR) region within the NCR domain. Further characterization of this STR region included analyses of deletion mutants and N2ICΔOP molecules containing point mutations; these studies indicate that phosphorylation of Ser2078 is a significant factor in the regulation of Notch2 function in this system.DISCUSSIONThe Notch signaling pathway participates in many cell fate decisions during embryonic and adult tissue development (for reviews see Refs. 1Egan S.E. St Pierre B. Leow C.C. Curr. Top. Microbiol. Immunol. 1998; 228: 273-324Crossref PubMed Google Scholar, 46Robey E. Curr. Opin. Genet. Dev. 1997; 7: 551-557Crossref PubMed Scopus (108) Google Scholar, 47Milner L.A. Bigas A. Blood. 1999; 93: 2431-2448Crossref PubMed Google Scholar). The regulation of such a widely used pathway is complex and involves different mechanisms. Phosphorylation of the Notch molecule was first described in Drosophila (35Kidd S. Lieber T. Young M.W. Genes Dev. 1998; 12: 3728-3740Crossref PubMed Scopus (192) Google Scholar) and has more recently been reported in mammalian cells (39Redmond L. Oh S.R. Hicks C. Weinmaster G. Ghosh A. Nat. Neurosci. 2000; 3: 30-40Crossref PubMed Scopus (278) Google Scholar, 40Shimizu K. Chiba S. Hosoya N. Kumano K. Saito T. Kurokawa M. Kanda Y. Hamada Y. Hirai H. Mol. Cell. Biol. 2000; 20: 6913-6922Crossref PubMed Scopus (141) Google Scholar). Here we have presented evidence that phosphorylation is important in the regulation of Notch2 activity in the context of specific cytokine signals.Intracellular Notch1 and Notch2 have been shown to function as activated molecules in several systems. Although both homologs display similar biochemical activity when assayed in in vitrosystems (17Kato H. Sakai T. Tamura K. Minoguchi S. Shirayoshi Y. Hamada Y. Tsujimoto Y. Honjo T. FEBS Lett. 1996; 2–3: 221-224Crossref Scopus (89) Google Scholar, 18Ordentlich P. Lin A. Shen C.P. Blaumueller C. Matsuno K. Artavanis Tsakonas S. Kadesch T. Mol. Cell. Biol. 1998; 18: 2230-2239Crossref PubMed Scopus (230) Google Scholar), when overexpressed in hematopoietic cells they have been shown to exhibit functional specificity (25Bigas A. Martin D.I. Milner L.A. Mol. Cell. Biol. 1998; 18: 2324-2333Crossref PubMed Scopus (183) Google Scholar). We have now demonstrated that deletion of a STR region between amino acids 2067–2099 abrogates the cytokine-restricted activity of Notch2 on 32D myeloid differentiation. Within this region, we have determined that phosphorylation of the Ser2078 residue in the context of G-CSF stimulation is important in regulating N2ICΔOPactivity.Notch2 Function Is Regulated by PhosphorylationRegulation of Notch activity occurs at a variety of different levels during the protein lifespan. Prior to placement on the cell surface, the Notch protein undergoes proteolytic processing to generate a heterodimeric receptor (48Blaumueller C.M. Qi H. Zagouras P. Artavanis Tsakonas S. Cell. 1997; 90: 281-291Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar) and glycosylation by fringe to modulate ligand binding specificity (38Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (335) Google Scholar). Following ligand binding, Notch is cleaved by at least 2 different proteases, γ-secretase/presenilins (49Song W. Nadeau P. Yuan M. Yang X. Shen J. Yankner B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6959-6963Crossref PubMed Scopus (310) Google Scholar, 50Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat. Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (357) Google Scholar) and disintegrin-metalloproteases (ADAM) (51Sotillos S. Roch F. Campuzano S. Development. 1997; 124: 4769-4779PubMed Google Scholar, 52Brou C. Logeat F. Gupta N. Bessia C. LeBail O. Doedens J.R. Cumano A. Roux P. Black R.A. Israel A. Mol. Cell. 2000; 5: 207-216Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar), to release the intracellular domain. Finally, intracellular Notch is modified by phosphorylation in response to ligand binding (40Shimizu K. Chiba S. Hosoya N. Kumano K. Saito T. Kurokawa M. Kanda Y. Hamada Y. Hirai H. Mol. Cell. Biol. 2000; 20: 6913-6922Crossref PubMed Scopus (141) Google Scholar) and to differentiation signals (this report).It has been reported that phosphorylation of DrosophilaNotch is necessary for nuclear translocation and interaction with the transcription factor Suppressor of Hairless (Su(H)) (35Kidd S. Lieber T. Young M.W. Genes Dev. 1998; 12: 3728-3740Crossref PubMed Scopus (192) Google Scholar). Several recent studies also support a role for phosphorylation in the regulation of subcellular localization and activity of intracellular Notch. For example, Notch-IC proteins have been found in the nucleus of dendritic cells in distinct phosphorylated forms (39Redmond L. Oh S.R. Hicks C. Weinmaster G. Ghosh A. Nat. Neurosci. 2000; 3: 30-40Crossref PubMed Scopus (278) Google Scholar). In other reports, hyperphosphorylation of Notch has been associated with Notch activation, nuclear translocation, and transformation ability (41Ronchini C. Capobianco A.J. Oncogene. 2000; 19: 3914-3924Crossref PubMed Scopus (39) Google Scholar, 40Shimizu K. Chiba S. Hosoya N. Kumano K. Saito T. Kurokawa M. Kanda Y. Hamada Y. Hirai H. Mol. Cell. Biol. 2000; 20: 6913-6922Crossref PubMed Scopus (141) Google Scholar). We previously observed that N2ICΔOP molecule is able to inhibit differentiation when induced by GM-CSF but fails to block differentiation in the presence of G-CSF (25Bigas A. Martin D.I. Milner L.A. Mol. Cell. Biol. 1998; 18: 2324-2333Crossref PubMed Scopus (183) Google Scholar). In the present study, we have found that N2ICΔOP is phosphorylated in 32D cells in IL3 or GM-CSF medium and increased phosphorylation of N2ICΔOP occurs when cells are incubated in G-CSF. Together these findings suggest the possibility that G-CSF induces specific phosphorylation of N2ICΔOP, thereby rendering the molecule inactive. By contrast, when cells are incubated with G-CSF, N1ICΔOP molecule decreased its phosphorylation suggesting that both molecules may be differentially regulated in response to the same cytokine.Our results show increased phosphorylation of at least three spots (A, B, and C) in the N2IC and N2ICΔOP after G-CSF stimulation. In all three mutants that are able to block G-CSF-induced differentiation peptide A is absent, thus strongly suggesting that Ser2078 is critical in regulating the Notch2 cytokine specific activity in 32D cells. The fact that peptide A is completely absent in the deletion mutant Δ2077–2081, the double point mutant S2078A/S2090A, and the S2078A single mutant suggests that this peptide includes Ser2078. When a theoretical trypsin digest map for N2ICΔOP is generated, the peptide containing Ser2078 is 29 amino acids long (Leu-Leu-Asp-Glu-Tyr-Asn-Val-Thr-Pro-Ser-Pro-Pro-Gly-Thr-Val-Leu-Thr-Ser2078-Ala-Leu-Ser-Pro-Val-Leu-Cys-Gly-Pro-Asn-Arg) and its predicted mobility coincides with the actual migration of peptide A. We cannot exclude that spots B and C are multiple phosphorylation states of the same peptide or partial digestions containing Ser2078 thus explaining their decreased intensity in the tryptic maps lacking peptide A. Moreover, we have shown that different mutant molecules that lack Ser2078 are able to inhibit G-CSF induced differentiation. These results strongly suggest that phosphorylation of this residue is crucial for regulating Notch2 cytokine specific activity in 32D cells. Of note, Ser2078 is unique to Notch2.We have focused on phosphopeptides A, B, and C in this study as these were the only peptides to reproducibly correlate with Notch2 function and cytokine stimulus. However, in individual experiments, different peptides showed variable differences in intensity following exposure to G-CSF or GM-CSF. Thus, we cannot exclude the possibility that phosphorylation of other residues in the N2ICΔOP molecule may also influence its activity. It also remains possible that other residues in addition to Ser2078 contribute to the appearance or disappearance of phosphopeptide A. It is worth noting that initial phosphorylation of Ser2078 may be required to establish target motifs for subsequent phosphorylation by CKI (-S(P)-A-L-S-) or GSK3 (-T-V-L-T-S(P)-A-L-S). In this sense and using one of the available proteomics data bases (PhophoBase v2.0; Center for Biological Sequence Analysis; www.cbs.dtu.dk), we searched for potential phosphorylation sites in the N2ICΔOP sequence. This search revealed consensus sequences for calmodulin kinase II, (CaMKII), casein kinase I (CKI), casein kinase II (CKII), glycogen synthase kinase 3 (GSK3), protein kinase A (PKA), and protein kinase C (PKC). The specific kinase responsible for phosphorylation of Ser2078 remains to be determined and is a subject of current investigation. Molecular pathways that might directly link G-CSF signal transduction to phosphorylation of Notch2 are particularly intriguing. Many cytokines, including G-CSF and GM-CSF, activate kinase signaling cascades (53Suzuki K. Hino M. Hato F. Tatsumi N. Kitagawa S. Blood. 1999; 93: 341-349Crossref PubMed Google Scholar), some of which also interact with the Notch pathway. For example, interactions between the Notch and Ras/MAP kinase signaling pathways have been shown to result in reciprocal modulation of the two pathways: in some cases, Ras signaling is affected by Notch inhibition of MAP kinase activity (18Ordentlich P. Lin A. Shen C.P. Blaumueller C. Matsuno K. Artavanis Tsakonas S. Kadesch T. Mol. Cell. Biol. 1998; 18: 2230-2239Crossref PubMed Scopus (230) Google Scholar, 54Ikeya T. Hayashi S. Development. 1999; 126: 4455-4463Crossref PubMed Google Scholar, 55Zecchini V. Brennan K. Martinez-Arias A. Curr. Biol. 1999; 9: 460-469Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 56Berset T. Hoier E.F. Battu G. Canevascini S. Hajnal A. Science. 2001; 291: 1055-1058Crossref PubMed Scopus (218) Google Scholar); in others, Notch signaling is modulated by MAPK-induced phosphorylation of molecular components of the Notch pathway (reviewed in Ref. 1Egan S.E. St Pierre B. Leow C.C. Curr. Top. Microbiol. Immunol. 1998; 228: 273-324Crossref PubMed Google Scholar). MAPK consensus sequences are particularly ambiguous, but include a Thr/Ser residue followed by a Pro, which are also present in the STR of Notch1 and Notch2 (Fig. 1).A Role for Notch Homologs in Myeloid DifferentiationOver the past few years it has become clear that Notch signaling is involved in the regulation of hematopoiesis (reviewed in Ref. 47Milner L.A. Bigas A. Blood. 1999; 93: 2431-2448Crossref PubMed Google Scholar). Although many details are still lacking, the influence of Notch1 at multiple differentiation branch points during lymphoid development has been well documented. For example, Notch1 first influences the development of cells along the T or B cell lineage (23Pui J.C. Allman D. Xu L. DeRocco S. Karnell F.G. Bakkour S. Lee J.Y. Kadesch T. Hardy R.R. Aster J.C. Pear W.S. Immunity. 1999; 11: 299-308Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar) and then participates in the determination of sequential cell fates during T cell differentiation (22Robey E. Chang D. Itano A. Cado D. Alexander H. Lans D. Weinmaster G. Salmon P. Cell. 1996; 87: 483-492Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 57Washburn T. Schweighoffer E. Gridley T. Chang D. Fowlkes B.J. Cado D. Robey E. Cell. 1997; 88: 833-843Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 24Radtke F. Ferrero I. Wilson A. Lees R. Aguet M. MacDonald H.R. J. Exp. Med. 2000; 191: 1085-1094Crossref PubMed Scopus (127) Google Scholar).Definitive studies of Notch function in myeloid development (including erythroid, megakaryocytic, granulocytic, and monocytic lineages) have been more elusive, possibly because of the relative complexity of Notch signaling in this system. However, a number of studies indicate that Notch signaling is involved in myelopoiesis, and provide clues as to specific functions. Expression of active (Notch-IC) molecules or ligand-induced activation of Notch in myeloid cell lines such as 32D (11Milner L.A. Bigas A. Kopan R. Brashem-Stein C. Bernstein I.D. Martin D.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13014-13019Crossref PubMed Scopus (170) Google Scholar, 42Li L. Milner L.A. Deng Y. Iwata M. Banta A. Graf L. Marcovina S. Friedman C. Trask B.J. Hood L. Torok Storb B. Immunity. 1998; 8: 43-55Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) and HL60 (26Carlesso N. Aster J.C. Sklar J. Scadden D.T. Blood. 1999; 93: 838-848Crossref PubMed Google Scholar), or in primary hematopoietic cells (30Jones P. May G. Healy L. Brown J. Hoyne G. Delassus S. Enver T. Blood. 1998; 92: 1505-1511Crossref PubMed Google Scholar, 31Varnum-Finney B. Purton L.E. Yu M. Brashem-Stein C. Flowers D. Staats S. Moore K.A. Le Roux I. Mann R. Gray G. Artavanis-Tsakonas S. Bernstein I.D. Blood. 1998; 91: 4084-4091Crossref PubMed Google Scholar, 32Han W. Ye Q. Moore M.A. Blood. 2000; 95: 1616-1625Crossref PubMed Google Scholar) generally result in undifferentiated phenotypes. However, in both invertebrate and mammalian systems, the effects of Notch have been shown to be both multifaceted and highly context-dependent. Thus, depending on the specific cell type and environmental signals, Notch activity may promote or inhibit differentiation, prevent or induce apoptosis (58Jehn B.M. Bielke W. Pear W.S. Osborne B.A. J. Immunol. 1999; 162: 635-638PubMed Google Scholar, 59Ohishi K. Varnum-Finney B. Flowers D. Anasetti C. Myerson D. Bernstein I.D. Blood. 2000; 95: 2847-2854Crossref PubMed Google Scholar), and promote cell cycle progression and mitosis or induce cell cycle arrest (60Baonza A. Garcia-Bellido A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2609-2614Crossref PubMed Scopus (78) Google Scholar, 61Johnston L.A. Edgar B.A. Nature. 1998; 6688: 82-84Crossref Scopus (225) Google Scholar). In the hematopoietic system, context-dependent effects of Notch have been observed in progenitor populations (62Varnum-Finney B. Xu L. Brashem-Stein C. Nourigat C. Flowers D. Bakkour S. Pear W.S. Bernstein I.D. Nat. Med. 2000; 6: 1278-1281Crossref PubMed Scopus (525) Google Scholar), and during lineage differentiation of T cells (reviewed in Ref. 63Osborne B. Miele L. Immunity. 1999; 11: 653-663Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), monocytes (59Ohishi K. Varnum-Finney B. Flowers D. Anasetti C. Myerson D. Bernstein I.D. Blood. 2000; 95: 2847-2854Crossref PubMed Google Scholar), myeloid (25Bigas A. Martin D.I. Milner L.A. Mol. Cell. Biol. 1998; 18: 2324-2333Crossref PubMed Scopus (183) Google Scholar), and erythroid cells (28Lam L.T. Ronchini C. Norton J. Capobianco A.J. Bresnick E.H. J. Biol. 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