Cytoplasmic Signaling Circuitry Programs Many of the Traits of Cancer Chapter 6 Cytoplasmic Signaling Circuitry Programs Many of the Traits of Cancer 5.9 - 6.2 ~ 6.9 - Apr 3 & 12, 2007

5.9 The Ras protein functions as a G protein - The ras oncogene triggers many of the same changes in cells which are transformed by erbB (truncated EGF-R) or sis (PDGF-B). - Could Ras be found somewhere downstream of erbB and sis ? - Do the signals emitted by EGF-R and PDGF-R converge on some common molecule ?

growth factor receptor Ras GRB2: growth factor receptor- bound protein 2 SH2, SH3: Src homolog 2 or 3 Sos: son of sevenless (guanosine diphosphate) (guanosine triphosphate)

Regulation of Ras (a GTPase) activity (mitogenic signals) Guanine nucleotide Exchange Factor e.g., Sos, which catalyzes conversion of inactive GDP-bound Ras to the active GTP-bound form. GTPase- Activating (or Accelerating) Protein

Regulation of Ras (a GTPase) activity (mitogenic signals) Guanine nucleotide Exchange Factor e.g., Sos, which catalyzes conversion of inactive GDP-bound Ras to the active GTP-bound form. GTPase- Activating (or Accelerating) Protein (e.g., H-Ras)

The structure of the Ras protein Two most frequently altered a.a. residues found in human oncoproteins are glycine 12 and glutamine 61, which are closed associated with the γ-phosphate of GTP. Figure 5.31 The Biology of Cancer (© Garland Science 2007)

EGF/EGFR-mediated Ras activation

Two classes of GTPase switch proteins 1. trimeric (large) G proteins, (guanine nucleotide-binding proteins) : 1. trimeric (large) G proteins, which directly bind to and activated by receptors, e.g., Smoothened, Frizzled.

2. monomeric (small) G proteins, such as Ras and various Ras- like proteins. Ras doesn’t bind to receptors. It indirectly links to receptors via adapter proteins (such as GRB2) and GEF proteins (such as Sos).

6.2 The Ras protein stands in the middle of a complex signaling cascade The story of son of sevenless (sos) the genetics of eye development in Drosophila In the absence of sevenless, the 7th cell in each ommatidium fail to form. Figure 6.5 The Biology of Cancer (© Garland Science 2007)

6.3 Tyrosine phosphorylation controls the location and thereby the actions of many cytoplasmic signaling proteins Why is phosphorylation critical? - protein relocalization.

Domain structure of Src protein substrate recognition - proline rich (SH3 domain) - p-tyrosine (SH2 domain) catalytic SH1 domain Figure 6.7a The Biology of Cancer (© Garland Science 2007)

Structure and function of SH2 domain act as a modular plug Figure 6.8a The Biology of Cancer (© Garland Science 2007)

Signal-transducing proteins attach to phosphorylated receptors a phosphotyrosine phosphatase (PTP) Figure 6.9 The Biology of Cancer (© Garland Science 2007)

Signaling molecules with SH2 and SH3 domains Figure 6.10a The Biology of Cancer (© Garland Science 2007)

Molecular “ligand domains” and “receptor domains” in order to facilitate intermolecular interactions Figure 6.10b The Biology of Cancer (© Garland Science 2007)

Table 6.2 The Biology of Cancer (© Garland Science 2007)

6.4 SH2 groups explain how growth factors activate Ras and acquire signaling Figure 6.12 The Biology of Cancer (© Garland Science 2007)

Mammalian Ras proteins have been studied in great detail because mutants Ras proteins are associated with many types of human cancer. These mutant proteins, which bind but cannot hydrolyze GTP, are permanently in the “on” state and contribute to neoplastic transformation. Most oncogenic, constitutively active Ras protein contain a mutation at position 12. Replacement of the normal glycine-12 with other amino acid blocks the functional binding of GAP, and in essence “lock” Ras in the active GTP-bound state.

Sidebar 6.3 The SH2 domain of Src has two alternative functions inactive dephosphorylation of Y527 phosphorylation of Y416 SH2/SH3 bind to p-receptor exposure of the catalytic cleft Figure 6.11 The Biology of Cancer (© Garland Science 2007)

6.5 A cascade of kinases forms one of three important signaling pathways downstream of Ras Figure 6.14 The Biology of Cancer (© Garland Science 2007)

The Ras effector loop can bind Raf, PI3K and Ral-GEF Figure 6.13 The Biology of Cancer (© Garland Science 2007)

Ras → Raf → MAP (Mitogen-activated protein) kinase pathway Raf, MEK and Erk1/2 are serine/threonine kinases phosphotyrosines attract signaling partners → relocalization of partners phosphorylation of serines/threonines → shift in structure → functional activation MEK: MAP/Erk kinase Erk: extracellular signal- regulated kinases 1 and 2 (Erk1/2) Figure 6.14 The Biology of Cancer (© Garland Science 2007)

Ras/Raf/MAP Kinase Pathway dimer Figure 6.10b The Biology of Cancer (© Garland Science 2007)

Activated MAPK induces gene transcription transcription factor TCF: ternary complex factor SRE: serum response element transcription factor SRF: serum response factor (early response gene) → enter the cell cycle

The Ras-PI3 kinase (PI3K) pathway (cytoplasm) Figure 6.15 The Biology of Cancer (© Garland Science 2007)

Generation of the 2nd messengers – DAG and IP3

PI3K converts PIP2 into PIP3 PI3K can bind to phosphotyrosine and Ras. Figure 6.16b The Biology of Cancer (© Garland Science 2007)

Docking of PH domains of Akt/PKB to PIP3 PTEN: phosphatase and tensin homolog PH domain: pleckstrin homology Figure 6.19a The Biology of Cancer (© Garland Science 2007)

Table 6.3 The Biology of Cancer (© Garland Science 2007)

AKT/PKB enhances the growth of cells Normal islet cells Islet cells expressing constitutively active Akt/PKB. The cells are fourfold larger than normal. Figure 6.20 The Biology of Cancer (© Garland Science 2007)

Table 6.4 The Biology of Cancer (© Garland Science 2007)

Sidebar 6.4 Ras is the prototype of a large family of similar proteins Small G proteins – 35 similarly structured proteins e.g., Ras, Ral, Rac, Ran, Rho… - All small G proteins operate like a binary switch, using a GTP-GDP-GTP cycle to flip back and forth between an on and an off state. - Each small G protein has its own specialized guanine nucleotide exchange factor (GEF) to activate it and its own GTPase-activating proteins (GAP)

6.7 A third Ras-regulated pathway acts through Ral, a distinct cousin of Ras (Ral guanine nucleotide exchange factors) (Ral-A and Ral-B are Ras-like proteins) (Rho family proteins) (control of cytoskeleton) play key roles in the motility that enables cancer cells to invade and metastasize Figure 6.21 The Biology of Cancer (© Garland Science 2007)

6.8 The Jak-STAT pathway allows signals to be transmitted from the plasma membrane directly to the nucleus cytokines Figure 6.22 The Biology of Cancer (© Garland Science 2007)

6.9 Cell adhesion receptors emit signals that converge with those released by growth factor receptors Figure 6.24a The Biology of Cancer (© Garland Science 2007)

Figure 6.24b The Biology of Cancer (© Garland Science 2007)