Question

Classes 7-8 Cytoplasmic Growth Signaling Be able to describe Ras structure and function. What kind of protein is it? How is it regulated (Fig. 5.30)? How many forms of Ras do humans express? Be able to describe how Ras interacts with multiple downstream partners. What mechanisms render ras oncogenic? Also, how does Ras become oncogenic in the absence of ras mutations? Be able to describe how Ras interacts with the growth factor receptor signaling machinery. How did fly genetics help to make this link? (What is "sevenless" and "sos" )? Likewise, be able to describe the modular domain concept of protein structure, and how it relates to cellular signaling specificity. What are SH1, SH2 and SH3 domains? From what proto-oncogenic protein did they derive their name, and how do they relate to growth factor-ras signaling (cf Fig. 6.12)? What are "adaptor proteins"? Be able to describe how the structure of SH2 domains imparts specificity to tyrosine kinase signaling. Be able to name and describe the three Ras signaling pathways and what cognate "master control" proteins Ras interacts with to regulate these pathways: MAPK, P13K, Ral-GEF Likewise, describe how activated Ras (or growth factor signaling) exerts pleiotropic effects on cells by virtue of its relationship to these pathways. Be able to describe the P13K signaling pathway and what it regulates. Include in your description, PIP2, PIP3, PH domains (and what they do), PDK1, Akt/PKB and PTEN. Be able to name three downstream targets of the Akt kinase; what are the effects of each phosphorylation event on these targets with respect to biological response (cf Fig. 6.15). What kind of protein kinase is Akt? How does it differ from Src? Map, with arrows, the route from growth factor binding to Akt-dependent activation of its targets; make sure to include all participating proteins in the signal transduction pathway.

Answer 1

Ras is an intracellular monomeric GTPase switch protein and functions in transducing signals from many different RTks. Ras cycles between an inactive GDP-bound form and active GTP-bound form. Ras cycling requires the assistance of two proteins, GEF (Guanine nucleotide exchange factor) and GAP (GTPase activating protein). Ras activation is accelerated by a protein called GEF, which binds to the Ras-GDP complex, causing dissociation of the bound GDP. Because GTP is present in cells at a higher concentration than GDP, GTP binds spontaneously to empty Ras molecules, with the release of GEF. The average lifetime of a GTP bound to Ras is about 1 minute. GAP, which binds to Ras-GTPand accelerates its intrinsic GTPase activity. Binding of a ligand to an RTK lead to activation of Ras. Two cytosolic proteins, an adaptor protein, and GEF provide the key links between RTK and Ras. Adaptor proteins bind to an activated receptor, and then other proteins bind to them, and may, therefore, become substrates for the receptor. Adaptor has no catalytic activity of its own but serves merely to bring other proteins to the receptor. The cytosolic adaptor protein GRB2 binds to a specific phosphotyrosine on an activated, ligand bound receptor and to the cytosolic GEF, Sos protein. Sos protein then promotes the formation of active Ras-GTP. An SRc Homology domain, in GRB2, binds to a specific phosphotyrosine residue in the activated receptor.  

MAPK pathway:

GGTPU Activated has protein plasma membrane Raf cytosol Selther phosphaylation. Mek) Tyr/ the phosphorylation. MAP-kinase sol the phosphorylation. Changes in Protein activity changes in gene expression. Pathway. MAP- Kinase

1. Growth factor binds to its receptor on the surface of the cell.

2. Receptor subunits cluster together and allow autophosphorylation.

3. An adapter protein complex (GRB2/SOS) binds to the phosphorylated form of the receptor and allows activation of Ras (exchange of GDP on Ras for GTP).

4. Ras-GTP phosphorylates Raf (MAPKKK)

5. Phosphorylated Raf activates MEK (MAPKK) by phosphorylation.

6. phosphorylated MEK activates MAPK (ERK) by phosphorylation.

7. phosphorylated MAPK (ERK) translocates into the nucleus.

8. In the nucleus phosphorylated MAPK (ERK) activate transcription factors by phosphorylation.

9. Activated transcription factors allow the expression of genes involved in cellular proliferation and passage through the cell cycle.

10. Cell growth and mitosis.