Determination of ERK Activity: Anti-phospho-ERK Antibodies and In Vitro Phosphorylation

Abstract

The ERK signaling cascade is composed of several protein kinases that sequentially activate each other by phosphorylation. This pathway is a central component of a complex signaling network that regulates important cellular processes including proliferation, differentiation, and survival. In most of these cases, the ERK cascade is activated downstream of the small GTPase Ras that, upon activation, recruits and activates the first tier in the cascade, which contains the Raf kinases. Afterward the signal is further transmitted by MEKs, ERKs, and often RSKs in the MAPKK, MAPK, and MAPKAPKs tiers of the cascade, respectively. ERKs and RSKs can further disseminate the signal by phosphorylating and modulating the activity of a large number of regulatory proteins including transcription factors and chromatin modifying enzymes. Understanding the mechanisms of activation and the regulation of the various components of this cascade will enhance our insight into the regulation of the ERK-dependent cellular processes in normal cells or of their malfunctioning in various diseases, including cancer. In this chapter, we describe methods used to determine the activity of ERKs, which upon slight modifications can also be used for the study of other signaling kinases, either within the cascade or in other pathways. These methods have been successfully applied to study the ERK signaling cascades in a variety of tissue-cultured cell lines, homo-genized animal organs, and lower organisms. As such, the use of these methods should expand our knowledge on the regulation of many distinct systems and upon induction of various stimulations.

WNT‐5A triggers Cdc42 activation leading to an ERK1/2 dependent decrease in MMP9 activity and invasive migration of breast cancer cells

“…The phosphorylation of ERK1/2 were analyzed after 1 h, 6 h, 12 h, and 24 h by Western blotting using a polyclonal antibody specific to phosphorylated sites at Thr202/Tyr204. Phosphorylation of these sites is coupled to the activity of ERK1/2 and thus a reliable and indirect method for estimating ERK1/2 activity (Osabe and Negishi, 2011; Procaccia et al., 2010). Based on this, we have from here onwards referred to our determinations of phosphor‐ERK1/2 levels as ERK1/2 activities.Both breast cancer cell lines had the same activation kinetics of ERK1/2 when stimulated with rWNT‐5A.…”

Boron‐Based Inhibitors of Acyl Protein Thioesterases 1 and 2

“…A) Compound 4 inhibited N‐Ras‐induced phosphorylation of ERK. MDCK/F3 cells were treated with the indicated concentrations of palmostatin B or compound 4 for 6 h. The cell lysates were prepared and subjected to Western blotting with anti‐phospho‐ERK and anti‐ERK antibodies 16. The intensity of each band was measured with ImageJ (http://rsbweb.nih.gov/ij/).…”

“…An equal amount of proteins was loaded onto a 10 % SDS‐PAGE gel. The phospho‐ERK1/2 and ERK1/2 were detected with anti‐phospho‐ERK and anti‐ERK antibodies, respectively 16…”

Inhibition of human breast cancer Matrigel invasion by Streptolysin O activation of the EGF receptor ErbB1

“…Downstream of ErbB1 is the Raf-MEK-ERK kinase pathway that transmits intracellular signals by phosphorylation of transcription factors and enzymes [14, 15]. ERK becomes activated by MEK-catalyzed dual phosphorylation of Thr-X-Tyr in the kinase activation loop [16]. We used phosphosite-specific antibodies to compare ERK activation in MDA-MB-231 cells with and without addition of SLO (Figure 4).…”