Presentation Type

Poster

Faculty Mentor’s Full Name

Mark Grimes

Faculty Mentor’s Department

Division of Biological Sciences

Abstract

SRC-family kinases (SFKs) are important in regulating cell-fate in neuroblastoma cells. PAG1, binds SFKs and helps regulate their activity by directing their movement within the cell. To better understand the role of PAG1, we created a line of mutant SH-SY5Y cells, called PAG1TM- cells, which express a truncated PAG1 protein, lacking a transmembrane domain. These cells still express high levels of mutant, cytosolic PAG1. We have found that PAG1TM- cells express a hyper-cancerous phenotype, including increased proliferation, and display higher global levels of active phosphorylated SFKs (pSFKs). The activity of pSFKs in PAG1TM- is still unknown. Protease protection experiments suggest that PAG1TM- cells may accumulate more pSFKs in multivesicular bodies (MVBs), however fractionation experiments have indicated that pSFKs primarily localize to early endosome and cytosol fractions. Once in MVBs, proteins may be recycled back to the plasma membrane or targeted to lysosomes for degradation. Alternatively, MVB cargo may be incorporated into exosomes and released by fusion of MVBs with the plasma membrane. SH-SY5Y cells, have been shown to release exosomes containing SFKs, including FYN and LYN. To determine if PAG1 is important for trafficking of SFKs into exosomes, I isolated exosomes from both wild-type (WT) and PAG1TM- SH-SY5Y cells and probed digested exosomes for SFKs. I found both LYN and pSFKs were greatly reduced in exosomes from PAG1TM- cells.

Category

Life Sciences

Share

COinS
 
Apr 17th, 11:00 AM Apr 17th, 12:00 PM

Effect of PAG1 on Src-Family Kinase Trafficking in Neuroblastoma Exosomes

UC South Ballroom

SRC-family kinases (SFKs) are important in regulating cell-fate in neuroblastoma cells. PAG1, binds SFKs and helps regulate their activity by directing their movement within the cell. To better understand the role of PAG1, we created a line of mutant SH-SY5Y cells, called PAG1TM- cells, which express a truncated PAG1 protein, lacking a transmembrane domain. These cells still express high levels of mutant, cytosolic PAG1. We have found that PAG1TM- cells express a hyper-cancerous phenotype, including increased proliferation, and display higher global levels of active phosphorylated SFKs (pSFKs). The activity of pSFKs in PAG1TM- is still unknown. Protease protection experiments suggest that PAG1TM- cells may accumulate more pSFKs in multivesicular bodies (MVBs), however fractionation experiments have indicated that pSFKs primarily localize to early endosome and cytosol fractions. Once in MVBs, proteins may be recycled back to the plasma membrane or targeted to lysosomes for degradation. Alternatively, MVB cargo may be incorporated into exosomes and released by fusion of MVBs with the plasma membrane. SH-SY5Y cells, have been shown to release exosomes containing SFKs, including FYN and LYN. To determine if PAG1 is important for trafficking of SFKs into exosomes, I isolated exosomes from both wild-type (WT) and PAG1TM- SH-SY5Y cells and probed digested exosomes for SFKs. I found both LYN and pSFKs were greatly reduced in exosomes from PAG1TM- cells.