? Members of the mitogen activated protein kinase (MAPK) family are crucial for the accurate transmission of environmental and developmental signals in cells. Determining how these signaling proteins function is of fundamental importance for understanding both normal and abnormal cellular proliferation, differentiation, and programmed cell death, as occur in lung diseases. Recently, we identified ERK8 as a new member of this family that is preferentially expressed in human lung. Preliminary data suggest that ERK8 is expressed in bronchial epithelial and smooth muscle cells, and that ERK8 functions as a negative regulator of airway cell cycle progression. In this proposal, we will characterize the mechanisms regulating ERK8 activation, expression, and function in airway cells, focusing on interactions with other cellular proteins. To achieve this, we propose the following Specific Aims: ? ? Specific Aim 1. Determine the mechanism by which Src and MAPK kinases activate ERK8. Since c-Src has been shown to interact with and activate ERK8, we will identify specific domain(s) of ERK8 that is/are involved in this interaction using deletion and point mutations of ERK8. We will determine whether c-Src is required or sufficient for ERK8 activation. We will also test the requirement and sufficiency of known MAPK kinases to activate ERK8 and determine whether LMO2 serves as a substrate for ERK8. ? ? Specific Aim 2. Determine the mechanism by which ERK8 expression is regulated. Since ERK8 expression is tightly regulated, we will determine the half-life of endogenous and ectopically expressed wild type and mutant ERK8, whether ERK8 is poly-ubiquitinated, and whether specific proteasome inhibitors prevent its degradation. Finally, chimeric and point mutations will be used to define specific region(s) of ERK8 that target it for degradation. ? ? Specific Aim 3. Determine the function of ERK8. We will perform in situ hybridization and immunohistochemistry on human lung tissue to identify cell types that express ERK8 message and protein, respectively. Whole cell populations of cultured epithelial, smooth muscle cells will be transduced with wild type and mutant forms of ERK8 using a murine leukemia virus/vesicular stomatitis virus pseudo typed retroviral vector and their effect on cell cycle progression, and growth will be determined. These studies should provide insight into the signaling mechanisms involved in normal cellular function, and therefore, a better understanding of lung diseases where these processes are disrupted. ? ?
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| Saelzler, Matthew P; Spackman, Christy C; Liu, Yuru et al. (2006) ERK8 down-regulates transactivation of the glucocorticoid receptor through Hic-5. J Biol Chem 281:16821-32 |
| Kuo, Wen-Liang; Duke, Crystal J; Abe, Mark K et al. (2004) ERK7 expression and kinase activity is regulated by the ubiquitin-proteosome pathway. J Biol Chem 279:23073-81 |
