Part:BBa_K3170026

Ras-Raf system

Feel the Ras-Raf signal and activate TEVp

• Ras proteins are GTPases that act as molecular switches that regulate signaling pathways for cell proliferation, survival, growth, migration, differentiation or cytoskeletal activity. The Ras protein converts the signal of the extracellular growth factor by cycling between the inactive GDP binding state and the active GTP binding state. The exchange of GTP with GDP on RAS is regulated by guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). Activated RAS (RAS-GTP) regulates a variety of cellular functions through effectors, including Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine nucleotide dissociation stimulator (RALGDS).
• Ras protein, molecular weight 21kD, is located inside the cell membrane. It consists of 188 or 189 amino acids, its first domain contains a highly conserved sequence of 85 amino acid residues, followed by a domain of 80 amino acid residues, the structure of p21Ras is slightly different, except for the 25 ends of K2Ras. Amino acids are classified into type A and type B due to different exons. The last four amino acids of the remaining Ras family members are Cys1862A2A2X2COOH sequences. There are four isoforms of Ras protein: H2Ras, N2Ras, K2Ras4A and K2Ras4B, which are the products of three genes, and K2Ras4A and K2Ras4B are the results of different splicing of the same gene. After synthesis, the Ras protein needs to undergo a series of processing modifications before it can be located inside the cell membrane. In the first step, a farnesyl group farnesyl is added to the Cys residue in the CAAX tetrapeptide structure at the carboxy terminus of Ras by farnesyltrans2ferase (FTase), followed by AAX residues from C The cleavage of the terminal is detached, and the farnesylation of Cys is carboxymethylated. Finally, the Ras protein is localized in the cell membrane to be biologically active. Interfering with the farnesylation of the Ras protein, the growth of tumors activated by the ras gene is inhibited. To date, the Ras/Raf pathway is the clearest signal transduction pathway. When GTP replaces GDP and binds to Ras, Ras is activated, and then activates the filament 2 threonine kinase cascade amplification effect, recruiting cytoplasmic Raf21 silk 2 threonine kinase to the cell membrane, and Raf kinase phosphorylates MAPK kinase (MAPKK, Also known as MEK), MAPKK activates MAPK (MAPK also known as ERK). After MAPK is activated, it is transferred to the nucleus and directly activates the transcription factor.

Experimental Results

• Imaging begins approximately 2 hours after the media is replaced with a fluorescent imaging medium. Get images every 1 hour. SW1990 cells were observed using 440 nm excitation light, and the cells turned from cyan to yellow.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1589
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2612
  Illegal SapI.rc site found at 319