Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Distribution of ERK1/2 and ERK3 during normal rat fetal lung development


The extracellular regulated kinases-1 and -2 (ERK1/2) are well-characterized mitogen-activated protein kinases (MAPK) that play critical roles in proliferation and differentiation, whereas the function(s) of MAPK ERK3 are currently unknown. To understand better the roles of these kinases in development, the temporal distribution of ERK1, -2, and -3 proteins were investigated in multiple tissues. The ERK3 protein, in contrast to ERK1/2 varied both between and within individual organs over time. To characterize this variability in greater detail, the temporal and spatial distributions of activated ERK1/2 and ERK3 during rat fetal lung development were investigated. The diphosphorylated (activated) forms of ERK1/2 (dp-ERK1/2), ERK3, and its phosphorylated form (P-ERK3) decreased from embryonic day 17 (E17) through E21 while both ERK1 and ERK2 total proteins remained unchanged, indicating that ERK1/2 and ERK3 proteins are expressed independently during fetal lung development. In addition, characterization of the distribution of these proteins by fluorescent immunohistochemistry indicated that phosphorylated ERK1/2 and total ERK1/2 were distributed throughout multiple cell types, with the phosphorylated ERK1/2 colocalizing with prophase mitotic cells. In contrast, ERK3 was restricted to the distal lung epithelium during the pseudoglandular phase (E17) but shifted to the proximal airways, particularly Clara cells during the saccular stage (E21). The P-ERK3 colocalized with the mitotic marker P-histone H3 in fetal lung and in NIH3T3 and HeLa cells, implicating a potential role for P-ERK3 in mitosis. Thus, expression of ERK1/2 and ERK3 and their phosphorylated forms are expressed independently and are temporally and spatially localized during fetal lung morphogenesis. These observations will facilitate detailed functional analysis of these kinases to assess their roles in pulmonary development and diseases.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1994) In: Jaansen K (ed) Current protocols in molecular biology. Wiley, New York, 10.12.11–10.12.21

  2. Bind E, Kleyner Y, Skowronska-Krawczyk D, Bien E, Dynlacht BD, Sanchez I (2004) A novel mechanism for mitogen-activated protein kinase localization. Mol Biol Cell 15:4457–4466

  3. Blatt EN, Yan XH, Wuerffel MK, Hamilos DL, Brody SL (1999) Forkhead transcription factor HFH-4 expression is temporally related to ciliogenesis. Am J Respir Cell Mol Biol 21:168–176

  4. Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, DePinho RA, Panayotatos N, Cobb MH, Yancopoulos GD (1991) ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65:663–675

  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  6. Cardoso WV (2001) Molecular regulation of lung development. Annu Rev Physiol 63:471–494

  7. Cheng M, Boulton TG, Cobb MH (1996a) ERK3 is a constitutively nuclear protein kinase. J Biol Chem 271:8951–8958

  8. Cheng M, Zhen E, Robinson MJ, Ebert D, Goldsmith E, Cobb MH (1996b) Characterization of a protein kinase that phosphorylates serine 189 of the mitogen-activated protein kinase homolog ERK3. J Biol Chem 271:12057–12062

  9. Corson LB, Yamanaka Y, Lai KM, Rossant J (2003) Spatial and temporal patterns of ERK signaling during mouse embryogenesis. Development 130:4527–4537

  10. Coulombe P, Rodier G, Pelletier S, Pellerin J, Meloche S (2003) Rapid turnover of extracellular signal-regulated kinase 3 by the ubiquitin-proteasome pathway defines a novel paradigm of mitogen-activated protein kinase regulation during cellular differentiation. Mol Cell Biol 23:4542–4558

  11. Daniely Y, Liao G, Dixon D, Linnoila RI, Lori A, Randell SH, Oren M, Jetten AM (2004) Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium. Am J Physiol Cell Physiol 287:C171–C181

  12. De Paepe ME, Rubin LP, Jude C, Lesieur-Brooks AM, Mills DR, Luks FI (2000) Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development. Am J Physiol Lung Cell Mol Physiol 279:L967–L976

  13. Del Riccio V, van Tuyl M, Post M (2004) Apoptosis in lung development and neonatal lung injury. Pediatr Res 55:183–189

  14. Fanucchi MV, Murphy ME, Buckpitt AR, Philpot RM, Plopper CG (1997) Pulmonary cytochrome P450 monooxygenase and Clara cell differentiation in mice. Am J Respir Cell Mol Biol 17:302–314

  15. Gabay L, Seger R, Shilo BZ (1997) MAP kinase in situ activation atlas during Drosophila embryogenesis. Development 124:3535–3541

  16. Garcia JI, Zalba G, Detera-Wadleigh SD, de Miguel C (1996) Isolation of a cDNA encoding the rat MAP-kinase homolog of human p63mapk. Mamm Genome 7:810–814

  17. Harper JW, Burton JL, Solomon MJ (2002) The anaphase-promoting complex: it’s not just for mitosis any more. Genes Dev 16:2179–2206

  18. Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD (1997) Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106:348–360

  19. Hilfer SR (1996) Morphogenesis of the lung: control of embryonic and fetal branching. Annu Rev Physiol 58:93–113

  20. Julien C, Coulombe P, Meloche S (2003) Nuclear export of ERK3 by a CRM1-dependent mechanism regulates its inhibitory action on cell cycle progression. J Biol Chem 278:42615–42624

  21. Khoor A, Gray ME, Singh G, Stahlman MT (1996) Ontogeny of Clara cell-specific protein and its mRNA: their association with neuroepithelial bodies in human fetal lung and in bronchopulmonary dysplasia. J Histochem Cytochem 44:1429–1438

  22. Kinane TB, Komatsuzaki K, Aleixo MD, Sunday ME, Ercolani L (1999) Regulation of the G protein Galphai2 by growth and development in fetal airway epithelium. Am J Respir Cell Mol Biol 20:35–42

  23. Kling DE, Narra V, Islam S, Kinane TB, Alessandrini A, Ercolani L, Donahoe PK, Schnitzer JJ (2001) Decreased mitogen activated protein kinase activities in congenital diaphragmatic hernia-associated pulmonary hypoplasia. J Pediatr Surg 36:1490–1496

  24. Kling DE, Lorenzo HK, Trbovich AM, Kinane TB, Donahoe PK, Schnitzer JJ (2002) MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs. Am J Physiol Lung Cell Mol Physiol 282:L370–L378

  25. Kling DE, Aidlen JT, Fisher JC, Kinane TB, Donahoe PK, Schnitzer JJ (2005) Nitrofen induces a redox-dependent apoptosis associated with increased p38 activity in P19 teratocarcinoma cells. Toxicol In Vitro 19:1–10

  26. Kyriakis JM (1995) Mitogen-activated protein kinase signaling cascades. In: Schleondorff D, Bonventre JV, (eds) Molecular nephrology: kidney function in health and disease. Marcel Dekker, New York pp 469–495

  27. Kyriakis JM (1999) Making the connection: coupling of stress-activated ERK/MAPK (extracellular-signal-regulated kinase/mitogen-activated protein kinase) core signalling modules to extracellular stimuli and biological responses. Biochem Soc Symp 64:29–48

  28. Liu Y, Stein E, Oliver T, Li Y, Brunken WJ, Koch M, Tessier-Lavigne M, Hogan BL (2004) Novel role for netrins in regulating epithelial behavior during lung branching morphogenesis. Curr Biol 14:897–905

  29. Marszalek A, Daa T, Kashima K, Nakayama I, Yokoyama S (2000) Ultrastructural and morphometric studies related to expression of the cell adhesion molecule PECAM-1/CD31 in developing rat lung. J Histochem Cytochem 48:1283–1289

  30. Meraldi P, Honda R, Nigg EA (2004) Aurora kinases link chromosome segregation and cell division to cancer susceptibility. Curr Opin Genet Dev 14:29–36

  31. Miettinen PJ, Warburton D, Bu D, Zhao JS, Berger JE, Minoo P, Koivisto T, Allen L, Dobbs L, Werb Z, Derynck R (1997) Impaired lung branching morphogenesis in the absence of functional EGF receptor. Dev Biol 186:224–236

  32. Morikawa Y, Fujii K, Okada T, Kiso Y, Sasaki F (1999) Quantitative changes of lung tissue components during perinatal period in rats. J Vet Med Sci 61:1229–1233

  33. Mulder KM (2000) Role of Ras and Mapks in TGFbeta signaling. Cytokine Growth Factor Rev 11:23–35

  34. Peters K, Werner S, Liao X, Wert S, Whitsett J, Williams L (1994) Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung. Embo J 13:3296–3301

  35. Ramirez MI, Millien G, Hinds A, Cao Y, Seldin DC, Williams MC (2003) T1alpha, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth. Dev Biol 256:61–72

  36. Rosner B (1990) Fundamentals of biostatistics, 3rd edn. PWS-Kent, Boston

  37. Roux PP, Blenis J (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68:320–344

  38. Schaeffer HJ, Weber MJ (1999) Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 19:2435–2444

  39. Schumacher S, Laass K, Kant S, Shi Y, Visel A, Gruber AD, Kotlyarov A, Gaestel M (2004) Scaffolding by ERK3 regulates MK5 in development. Embo J 23:4770–4779

  40. Seger R, Ahn NG, Boulton TG, Yancopoulos GD, Panayotatos N, Radziejewska E, Ericsson L, Bratlien RL, Cobb MH, Krebs EG (1991) Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation. Proc Natl Acad Sci USA 88:6142–6146

  41. Seternes OM, Mikalsen T, Johansen B, Michaelsen E, Armstrong CG, Morrice NA, Turgeon B, Meloche S, Moens U, Keyse SM (2004) Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway. Embo J 23:4780–4791

  42. Shannon JM, Hyatt BA (2004) Epithelial-mesenchymal interactions in the developing lung. Annu Rev Physiol 66:625–645

  43. Soloaga A, Thomson S, Wiggin GR, Rampersaud N, Dyson MH, Hazzalin CA, Mahadevan LC, Arthur JS (2003) MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of histone H3 and HMG-14. Embo J 22:2788–2797

  44. Souza P, Kuliszewski M, Wang J, Tseu I, Tanswell AK, Post M (1995) PDGF-AA and its receptor influence early lung branching via an epithelial-mesenchymal interaction. Development 121:2559–2567

  45. Tebar M, Boex JJ, Ten Have-Opbroek AA (2001) Functional overexpression of wild-type p53 correlates with alveolar cell differentiation in the developing human lung. Anat Rec 263:25–34

  46. Turgeon B, Saba-El-Leil MK, Meloche S (2000) Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa. Biochem J 346 Pt 1:169–175

  47. Warburton D, Schwarz M, Tefft D, Flores-Delgado G, Anderson KD, Cardoso WV (2000) The molecular basis of lung morphogenesis. Mech Dev 92:55–81

  48. Whitsett JA, Tichelaar JW (1999) Forkhead transcription factor HFH-4 and respiratory epithelial cell differentiation. Am J Respir Cell Mol Biol 21:153–154

  49. Williams MC, Cao Y, Hinds A, Rishi AK, Wetterwald A (1996) T1 alpha protein is developmentally regulated and expressed by alveolar type I cells, choroid plexus, and ciliary epithelia of adult rats. Am J Respir Cell Mol Biol 14:577–585

  50. Yamada T, Suzuki E, Gejyo F, Ushiki T (2002) Developmental changes in the structure of the rat fetal lung, with special reference to the airway smooth muscle and vasculature. Arch Histol Cytol 65:55–69

  51. Zecevic M, Catling AD, Eblen ST, Renzi L, Hittle JC, Yen TJ, Gorbsky GJ, Weber MJ (1998) Active MAP kinase in mitosis: localization at kinetochores and association with the motor protein CENP-E. J Cell Biol 142:1547–1558

  52. Zhou L, Lim L, Costa RH, Whitsett JA (1996) Thyroid transcription factor-1, hepatocyte nuclear factor-3beta, surfactant protein B, C, and Clara cell secretory protein in developing mouse lung. J Histochem Cytochem 44:1183–1193

  53. Zimmermann J, Lamerant N, Grossenbacher R, Furst P (2001) Proteasome- and p38-dependent regulation of ERK3 expression. J Biol Chem 276:10759–10766

Download references


We would like to thank Igor Bagayev for his confocal microscope expertise Antoinette Wetterwald (University of Bern) and Mary Williams (Boston University) for the kind gift of T1a antibodies, and Drucilla J. Roberts for reviewing the manuscript. This work was supported by the following grants from the National Heart, Lung, and Blood Institute HL069684 (DEK) and HL062615 (JJS) and National Institute of Child Health and Human Development P01HD039942 (JJS and PKD) of the National Institutes of Health.

Author information

Correspondence to David E. Kling.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kling, D.E., Brandon, K.L., Sollinger, C.A. et al. Distribution of ERK1/2 and ERK3 during normal rat fetal lung development. Anat Embryol 211, 139–153 (2006). https://doi.org/10.1007/s00429-005-0063-z

Download citation


  • Mitosis
  • MAP kinase
  • Cell differentiation
  • Lung development
  • Epithelial
  • Mesenchymal