Tuesday, March 12, 2013

Signal Transduction Pathway - MAP Kinase Pathways (Part 2)

In the last post, we have seen how the ERK pathway gets activated and how the Ras protein gets regulated. Here, we will proceed in continuation with previous post. We will start this post by the mode of Ras activation. The best mode would be that mediated by protein tyrosine kinases. There is autophosphorylation of protein tyrosine kinase receptors. This phosphorylation results in association with Ras guanine-nucleotide exchange factor (GEF). This is mediated by interaction with another protein that possesses the SH2 domain.
Confused? Okay! Let me make it easier to understand by taking an example. Sos is the protein which is a guanine nucleotide exchange factor and Grb2 is a protein in the cytosol which has the SH2 domain in unstimulated cells. The Ras is anchored to the inner leaflet of the plasma membrane with the help of lipids which are attached to the C-terminus of Ras. Now, the mechanism is: The Sos is bound to SH2 domain of Grb2 protein. When there is phosphorylation of the protein tyrosine kinase receptors, it creates a binding site for SH2 domain of Grb2 (as can be seen in the adjacent figure). This association of Grb2 with activated receptors localizes Sos also to the plasma membrane where it interacts with Ras protein. Sos then stimulates the guanine-nucleotide exchange of Ras from inactive Ras-GDP to active Ras-GTP. This active Ras-GTP complex then interacts with a number of proteins, including the Raf, mentioned in earlier post. As mentioned in the earlier post, Raf activates which then leads to activation of ERK.

Another very important role of ERK is the induction of immediate-early genes. So, what are immediate-early genes and how ERK induces? A fraction of activated ERK translocates to the nucleus. Activated ERK fraction regulates the transcription factors by phosphorylation which in turn is stimulated by induction of a family of approximately 100 genes which are called the immediate early genes. Lets take an example to make it more clear. The serum response factor (SRF) and Elk1 are transcription factors which bind to serum response element (SRE) in the promoter region of target sequence. Activated Erk (phosphorylated form) translocates to the nucleus where it phosphorylates and activates Elk1 (adjacent figure). Elk1 binds to SRE in a complex with SRF. Phosphorylation of Elk1 stimulate its activity as a transcriptional activator, as a result the immediate early genes are induced. There are many immediate early genes that themselves encode transcription factors and so there is induction in response to growth factor stimulation that leads to expression of array of downstream genes called secondary response genes. This ERK signalling stimulates cell proliferation.
The mammalian cells (and also yeast cells) have multiple MAPK pathways that control distinct cellular responses. Each cascade consists of three protein kinases: a terminal MAP kinase and two upstream kinases that regulate its activity. In mammalian cells, three major groups of MAP kinase have been identified. These include members of ERK family, JNK and p38 MAP kinase. The JNK and p38 MAP kinase cascade gets activated by members of Rho subfamily (small GTP binding protein) (which includes Cdc42, Rho and Rac) rather than by Ras. Also, the JNK and p38 MAPK leads to inflammation and cell death as against ERK kinases that lead to cell proliferation, survival and differentiation.
There are scaffold proteins which are associated with different components of each MAP kinase cascade as complexes. The specificity of each MAP kinase signalling is maintained in part by this organization in association with scaffold proteins. Example of JNK MAP kinase cascade. JIP-1 is a scaffold protein that organizes the JNK MAP kinase and its upstream molecules into a signalling cassette. Thus, JIP-1 binds MLK, MKK7 and JNK and organizes these components of JNK pathway into a signalling cassette. As can be seen in the diagram, Rac activates MLK which leads to specific and efficient activation of MKK7 which ultimately activates JNK. This interaction with scaffold proteins is thought to play an important role in determining the specificity of signalling pathways within the cell.