Lecture #11 | Identification of Cellular Oncogenes: Src

c-Src function

Links extracellular cues to intracellular signaling pathways

• able to activate downstream signaling pathways

• required for a normal cells activity but modulations can lead to the transformed phenotype

Protein structure of c-Src

Member of a family of tyrosine kinase

• Has two conserved, non catalytic domains that mediate protein-protein interactions

  • SH3: recognizes Pro-X-X-Pro

  • SH2: Binds P-Tyr

  • SH1: has a catalytic cite

  • Not found in just Src proteins, but first identified

• Myrsitic functional group that tells kinase to attach to inner plasma membrane

• Tyr at 527 position

Difference between v-Src gene and c-Src structure

v-Src gene lacks the C-terminal domain of c-Src, which contains Tyr at 527 position

SH2 domain

Binds phospho-tyrosine (yellow dots)

Why is v-Src more oncogenic that c-Src

C-terminal domain has a Tyr that is phosphorylated

• v-Src lacks phosphorylated Tyr

Phosphorylation of Tyr acts as an inhibitor signal (negative regulator)

Inactive conformation of c-Src

1. SH2 domain binds to phosphorylated Tyr 527 in C-terminus

2. proline linker region binds Sh3 domain, causes a folding in

   1. Tyr at active site is hidden

3. SRC cant phosphorylate substrates → cannot act as a kinase, folding makes inactive protein

How is c-src activated?

• De-phosphorylation of Y527.

• Other P-Y binding to the SH2 domain.

• Other Pro-X-X-Pro peptide binding to the SH3 domain.

• Auto-phosphorylation of Y416 in the activation loop.

  • Phosphorylation of Y416 relieves a steric barrier and allows the kinase to adopt a fully active form.

What happens if there is no Y527 like in v-Src?

No able to be inhibited, cannot be shut off and will always be regulating gene transcription

• keeps in creasing number of cells present

In addition to removing C-terminus, how else is c-Src activated?

Mutated Tyr 527 is actually a Phenylalanine

• lose of hydroxyl group that looses the cite of the phosphorylation

• so deletion of c-terminus or mutation of Tyr 527 can convert proton-oncogene to oncogene

Key Principle of Signal Transduction (ST) Pathways

• Nearly all kinases are actively regulated.

• They often have autoregulatory regions that are part of the same polypeptide or associated with other inhibitory proteins.

GF receptors

Growth factors bind to growth factor (GF) receptors and initiate the ST pathway, resulting in :

1. activation of immediate early genes

2. delayed early genes

What happens when growth factors bind to growth factor receptors?

GF receptors dimerize and autophosphorylate

• Proteins with SH2 domains phosphorylated growth factor receptors

• two together helps magnify and stabilize the response

c-Src under normal conditions

• activated by membrane-bound receptors in response to growth factors and other signals, including tyrosine and non-tyrosine kinase receptors.

  • Src binds via SH2 domains to P-Tyr on the receptor, disrupting the intramolecular inhibition (SH2–Tyr527 binding).

Targets of Src

• Proteins involved in signal transduction, cell cycle control, and cellular proliferation (e.g., IRS1, JAK, CDK1).

• Proteins involved in cell morphology and adhesion (e.g., cortactin).

• Transcription factors (e.g., estrogen and androgen receptor).

Ex: how cell morphology is altered by growth factors in the serum via c-Src

• Cell morphology is altered by growth factors in the serum via c-Src.

• Cortactin, an actin-binding protein, is a Src substrate; phosphorylation by Src promotes actin polymerization.

  

What happened in the 1980s with human tumors?

Oncogenes were identified directly from human tumors using transfections (introducing foreign DNA into mammalian cells).

NIH3T3 transformation/Foci assay

Method of identifying cellular oncogenes by introducing DNA from human tumors into normal mouse cells

• integration of tumor into mouse fibroblast

• Can create a library of viruses expressing DNA and see protein interactions