Signal transduction pathways mediate cell-cell interactions and integrate signals from the extracellular environment through specific receptors at the cell membrane. They play a pivotal role in regulating cellular growth and differentiation and in mediating many physiological and pathological processes, such as apoptosis, inflammation, and tumor development. The mitogenactivated protein kinases (MAPKs) constitute a cascade of phosphorylation events that transmit extracellular growth signals through membrane-boundRasto the nucleus of the cell. In this chapter, detailed protocols for analyzing the kinase activities of the key components of the MAPKs pathway—MEK1, ERK1, JNK, and p38 MAPK—are described. A brief introduction to the chemical inhibitors to the MAPKs pathway is provided in the method section of each kinase assay. Inhibitors of other signaling pathways are summarized in Table 1 . The reporter assay of cyclin D1, a key downstream target gene of MAPKs pathway, is also described in detail. Table 1. Signal Transduction Pathways and Inhibitors
|
Tyrosine Kinase Growth Factor Receptors |
||
|
HER2/Neu inhibitor |
Herceptin (anti-HER2 antibody) |
(53,54) |
|
EGF receptors |
IMC-C225 (anti-EGFR antidody) |
(55,56) |
|
EGFR-Tyrosine kinase |
ZD1839, pyridopyrimidines |
(54,57,58) |
|
RasSignaling |
||
|
Inhibitors ofRasfarnesyltransferase FPP analogues |
||
|
CAAX peptide analogues |
BZA-5B, L-739, Cys-4-ABA-Met FT1-276, SCH44342, SCH66336 |
(57–59) |
|
Bisubstrate inhibitors |
BMS-182878, BMS-184467 |
(57–59) |
|
Inhibitors of the Raf protein kinases |
ISIS5132, BAY43-9006 |
(57–59) |
|
Mitogen-Activated Protein Kinase Pathways |
||
|
Inhibitors of MEK |
PD184352, PD098059, U0126, R009-22110 |
(17,20,21,23–27) |
|
Inhibitors of ERK1 and ERK2 |
PD098059, E64D, calpeptin |
(5,28) |
|
Inhibitors of JNKs |
SB600125 |
(33,34) |
|
Inhibitors of p38 kinases |
SB203580, SB202190, SB242235, SB239063, SB220025, SB202474, SC68376, FR167653 |
(36,37,39–44,46) |
|
Inhibitors of PI3-Kinase Signaling Pathways |
Wortmannin, LY294002 |
(60,61) |
|
Protein Phosphatases Inhibitors Proteasome Inhibitors |
Microcystines, calyculins, cantharidin |
(67) |
|
Peptide aldehydes |
ALLN, MG132, PSI, MG115 |
(63) |
|
Peptide boronates |
MG262 |
(63) |
|
Nonpeptide inhibitors |
Lactacystin |
(63) |
|
DCI |
3,4-DCI |
(63,64) |
|
Peptide vinyl sulfones |
NLVS, YL3YS |
(63) |
|
Epoxyketones |
Epoxomicin, eponemycin, Ac-hFLFL-epoxide |
(63,65) |
|
Bivalent inhibitors |
Polyoxyethylene |
(66,67) |
|
Natural compound inhibitors |
TMC-95A, gliotoxin, EGCG |
(63,68) |
|
Histone Deacetylase inhibitors |
||
|
Short-chain fatty acids |
Butyrates |
(70,75) |
|
Hydroxamic acids |
Trichostatin A, oxamflatin |
(73) |
|
Cyclic peptides |
Trapoxin A, FR901228, apicidin |
|
|
Benzamides |
MS-27275 |
(78,80) |
|
SIR2 inhibitors |
Nicotinamide, splitomicin |
(78,79) |
