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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates
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HPIP and RUFY3 are noncanonical guanine nucleotide exchange factors of Rab5 to regulate endocytosis-coupled focal

Saratchandra Singh Khumukcham1, Vasudevarao Penugurti1, Suresh Bugide1

  • 1Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India.

The Journal of Biological Chemistry
|October 5, 2023
PubMed

Insights

Proto-oncoprotein hematopoietic PBX-interacting protein (HPIP) and RUN FYVE domain-containing protein 3 (RUFY3) regulate cell migration by activating Rab5. These proteins are crucial for focal adhesion disassembly and integrin trafficking.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Endocytosis is vital for cell migration, but its molecular regulators in focal adhesion turnover are not fully understood.
  • Proto-oncoprotein hematopoietic PBX-interacting protein (HPIP) and RUN FYVE domain-containing protein 3 (RUFY3) are implicated in cellular processes.

Purpose of the Study:

  • To elucidate the molecular mechanisms of HPIP and RUFY3 in endocytosis-coupled cell migration.
  • To identify the role of HPIP and RUFY3 in regulating Rab5 activity and focal adhesion turnover.

Main Methods:

  • Co-immunoprecipitation to assess protein interactions.
  • Site-directed mutagenesis to study HPIP coiled-coil domains.
  • Rab5 activation assays and cell migration assays.
  • Western blotting for FAK activation and integrin trafficking analysis.

Main Results:

  • HPIP and RUFY3 localize to focal adhesions and early endosomes with Rab5.
  • HPIP's coiled-coil domains are essential for binding Rab5 and RUFY3.
  • HPIP and RUFY3 function as noncanonical guanine nucleotide exchange factors, activating Rab5.
  • Loss of HPIP or RUFY3 impairs Rab5 activation, focal adhesion disassembly, FAK activation, and β1 integrin trafficking, hindering cell migration.

Conclusions:

  • HPIP and RUFY3 are critical noncanonical guanine nucleotide exchange factors for Rab5.
  • These proteins play a key role in integrin trafficking and focal adhesion turnover, essential processes for cell migration.
Keywords:
HPIP/PBXIP1RUFY3Rab5cell migrationendocytosisfocal adhesion kinase

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