REQUIREMENT OF THE DYNLRB FAMILY DYNEIN LIGHT CHAINS IN TRANSFORMING GROWTH FACTOR BETA SIGNALING
Open Access
- Author:
- Gao, Guofeng
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 20, 2006
- Committee Members:
- Sarah Bronson, Committee Chair/Co-Chair
Keith C Cheng, Committee Member
Mark Kester, Committee Member
Jiyue Zhu, Committee Member - Keywords:
- DYNLRB
dynein light chain
TGFbeta - Abstract:
- Transforming growth factor ? (TGF?) is the prototype for a superfamily of related members. TGF? family signaling controls various fundamental cellular functions, including cell proliferation and migration. Alterations in the TGF? signaling pathways have been implicated in a vast array of human cancers and other diseases as well. Despite advances in our understanding of TGF? signaling transduction, the mechanism of the multifunctional TGF? signaling is not completely clear yet. Therefore, further studies are required for deeper understanding of its diverse biological responses. DYNLRB1 was identified in the laboratory through a screen for TGF? receptor-interacting proteins, and it is also a dynein light chain. Dynein is molecular motor that plays many important functions in the cell. I hypothesized that DYNLRB family dynein light chains may play important specific functions in TGF? signaling. In this thesis, we investigated the regulation of the function of DYNLRB dynein light chains by TGF? and their role in TGF? signaling in mammalian cells and in primary zebrafish (Danio rerio) ovarian follicle cells. The experiments in Chapter 2 aimed to determine the involvement of DYNLRB1 in TGF? signaling and characterizing the function of DYNLRB1. The results from this chapter have demonstrated that the phosphorylation of DYNLRB1 on serine residues is stimulated by TGF? in Cos-1 cells and requires T?RII. It is further demonstrated that DYNLRB1 expression knockdown significantly impaired TGF?-induced fibronectin expression in MDCK cells. TGF?-induced DYNLRB1phosphorylation has been shown to be responsible for its recruitment to the dynein motor complex. Therefore, these results indicate a potential role for DYNLRB1 as a TGF? signaling intermediate, and its requirement in TGF? induction of fibronectin (a major component of the extracellular matrix), which plays important roles in cell adhesion, migration and differentiation. The experiments in Chapter 3 was designed to test the hypothesis that DYNLRB2 might be involved in Smad3-dependent TGF? signaling. Human DYNLRB2 is 77% identical to human DYNLRB1. Results in Chapter 3 have demonstrated that TGF? induction of SBE2-Luc, plasminogen activator inhibitor-1 expression in HaCaT cells and of Smad7-Luc in Hep3B cells, is significantly impaired, after blocking endogenous DYNLRB2 expression by siRNA. It is known that the induction of these genes is Smad3-dependent signaling event. However, similar blocking of DYNLRB2 expression does not inhibit the induction of ARE-Lux by TGF?, which has been demonstrated to be Smad2-dependent signaling event. Therefore, these results suggest that DYNLRB2 is specifically required in Smad3-dependent signaling. Further, it is demonstrated that TGF?-stimulated preferential interaction between DYNLRB2 and Smad3 may be the underlying mechanism for the requirement of DYNLRB2 in Smad3-dependent TGF? signaling. In addition, results have shown that TGF? stimulated a rapid recruitment of the DYNLRB2 to the dynein complex, and the TGF?-induced phosphorylation of DYNLRB2 is responsible for this TGF? stimulated recruitment. Collectively, results in this Chapter have demonstrated for the first time that DYNLRB2 is required for Smad3-dependent TGF? signaling. The experiments in Chapter 4 tested our hypothesis that in zebrafish ovarian follicle cells the function of zDYNLRB might be regulated by TGF?, and zDYNLRB might play an important role in TGF? signaling in such cells. It is shown that zDYNLRB is rapidly phosphorylated after TGF? stimulation, for which the T?RII is required. In addition, it is shown that the phosphorylation of zDYNLRB facilitates its rapid recruitment to the dynein complex, which is stimulated by TGF?. Knockdown experiments in zOFCs by morpholino have demonstrated that zDYNLRB was required for TGF? induction of TRE-Luc, 3TP-Lux and ARE-Lux. Thus, the results suggest a potential role for zDYNLRB in TGF? signaling in zebrafish ovarian follicle cells. Collectively, the experiments in this thesis demonstrated for the first time a requirement of DYNLRB2 dynein light chain in Smad3-dependent TGF? signaling in mammalian cells, and a requirement of DYNLRB1 as a potential TGF? signaling component in TGF? induction of fibronectin, respectively, as well as potential role for zDYNLRB in TGF? signaling in zebrafish ovarian follicle cells. However, the physiological significance of their functions needs to be addressed in vivo in the future.