Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Natalie G. Ahn

Second Advisor

Jingshi Shen

Third Advisor

Amy E. Palmer

Fourth Advisor

Marcelo C. Sousa

Fifth Advisor

Joseph J. Falke

Abstract

Approximately 70% human malignant melanomas contain oncogenic mutations in B-Raf, which constitutively activate this kinase, and result in constitutive activation of the B-RAF/MKK/ERK pathway. FAM129B, an 80 kDa polypeptide, was identified from a functional proteomics screen for phosphoproteins regulated by B-Raf signaling. FAM129B contains a Nterminal pleckstrin homology (PH) domain and 6 serines at the C-terminus, which can be phosphorylated under the regulation of B-Raf signaling pathway. FAM129B promotes cell invasion in 3D environment, in a manner of phosphorylation dependence. FAM129B PH domain is also important for cell motility. Meanwhile, our lab discovered a novel protein-organelle complex involved in rear-directed membrane contractility and cell movement, named as "Wnt5a receptor-actomyosin polarity (WRAMP) structure". FAM129B was identified as one component of this structure. The goal of this project is to understand mechanistically how FAM129B promotes cell invasion.

Initial PIP strips screening found that FAM129B binds to a rarely studied phosphoinositide—PtdIns(5)P. Further in vitro and in vivo studies show that this interaction is important for the association and function of FAM129B towards WRAMP structures. FAM129B is required for the formation of WRAMP structure, PtdIns(5)P and enzymes that generate PtdIns(5)P, PIKfyve and MTMR3 localize at or nearby FAM129B in WRAMP structures. Ablation of the two enzymes interferes with the ability of cell to form WRAMP structures, and decrease the presence of FAM129B at the formed WRAMP structures. The results together suggest that FAM129B mediates WRAMP structure formation through its binding recognition of PtdIns(5)P.

FAM129B was characterized also in other regulatory events important in cell migration and invasion by using WM239A cells. In vivo kinase assay and following mass spectrometry proves that FAM129B is a direct substrate of ERK2. Co-immunoprecipitation found no protein obviously binds to FAM129B with or without phosphorylation. Luciferase reporter assays shows that FAM129B doesn’t mediate β-catenin-dependent transcription in response to Wnt3a signaling. Several key proteins known for cell migration were tested, overall I found surface integrin β1 decrease by about 50% in FAM129B knockdown cells, while the total amount maintains the same, which suggests that FAM129B may promote cell invasion in part by modulating surface integrin β1.

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