Touch down PCR are derived to enhance primer binding in the annealing step.

TLS Online TPP Program

#Id: 9641

#Unit 4. Cell Communication and Cell Signaling

Downstream of Ras is a highly conserved cascade of three protein kinases-

Ras → Raf → MEK → MAP kinase

TLS Online TPP Program

#Id: 9642

#Unit 4. Cell Communication and Cell Signaling

In unstimulated cells, 

Raf is phosphorylated by several cytosolic kinases on two sites:

1. Serine/threonine kinase domain 

2. N-terminal autoinhibitory domain

TLS Online TPP Program

#Id: 9643

#Unit 4. Cell Communication and Cell Signaling

Molecular Handcuff 

Phosphorylation of Raf is recognize by protein 14-3-3 dimer, which locks the autoinhibitory domain in a position where it blocks and inhibits the kinase active site

TLS Online TPP Program

#Id: 9644

#Unit 4. Cell Communication and Cell Signaling

TLS Online TPP Program

#Id: 9645

#Unit 4. Cell Communication and Cell Signaling

Active Ras⋅GTP binds to the N-terminal autoinhibitory domain of Raf, altering its conformation and allowing the phosphate attached to this domain to be removed by cytosolic phosphatases, causing loss of 14-3-3 binding.

TLS Online TPP Program

#Id: 9646

#Unit 4. Cell Communication and Cell Signaling

Active Raf phosphorylates MEK on one or two serine residues in its activation loop, activating MEK kinase activity