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A signaling circuit must solve 2 problems:
1. Specificity - how can a protein exchange signals with the small subset of intended signaling partners
2. How can a protein acquire rapid access to their signaling pathways in the sea of thousands of proteins?
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Serum-starved experiment
- Immediate early genes: no de novo protein synthesis, transcribed less than an hour after serum administered.
- ex: transcription factors, secreted growth factors, cytoskeleton construction
Indicated protein structure, configuration or localization important (not protein concentration)
- Delayed early genes: require de novo protein synthesis (blocked by cyclohexamide), within an hour after expression of immediate early genes
- ex: reoganization of actin fibers, enhanced migratory capacity, survival signals blocking apoptosis
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How does the level of Myc serve as an indicator of the amount of mitogens present?
Both Myc mRNA and prtoein levels turn over rapidly when growth factors or other mitogens are removed.
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Sevenless experiment
Sevenless = gene mutation resulting in lack of formation of the seventh ommatidium in the eye of Drosophilia
Discovery of Sos, promote GTP binding to Ras.
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Ras signaling pathway
EGFR -> Shc -> Grb2 -> Sos -> Ras
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What is the purpose of the tyrosine kinase activity in the cytoplasmic domain of the receptors?
Receptor phosphorylationg alters the physical location of downstream signaling partners
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SH2 domain
binds to phosphorylated tyrosines -> allows a protein to physially interact with another protein containig phosphotyrosine
- [acts as an intracellular receptor
- ligand = peptide sequence containing a phosphorylated tyrosine and a flanking 3-6 amino acid peptide sequence]
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Examples of SH2
- phospholipase C
- PI3K
- phosphatases
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How do growth factors activate a variety of downstream signaling pathways?
each phosphotyrosine on the cytoplasmic tails of RTK has unique specificity for particular SH2 containing proteins which is dictated by the aminio acids flanking the tyrosine
ex: PDGF receptor attracts Src, PI3K, Ras-GAP, SHP2, phospholipase
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Examples of SH2 with pasma membrane associated proteins
Ras-Gap -> Ras, GTP hydrolysis
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SH3 domain
binds to proline rich amino acid sequences in partner protiens -> proteins become substrates for the Src kinase
acts in a similar fashion to SH2 domain to bind intracellular ligands
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Adaptor proteins
proteins like Grb2 and Shc that have no fncctional domains beyond SH2 and SH3 domains
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How does Ras signaling ultimately lead to cell transformation?
When Ras binds to GTP, its effector loop domain will physcially interact with several alternate downstream effector proteins (very poor affinity when Ras bound to GDP)
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