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Wednesday, October 21, 2020



The Ras–MAP kinase pathway

Ras is a small Gprotein that is constitutively associated with the plasma membrane and is frequently activated in response to diverse stimuli that promote cell division (Figure 7.10). It can exist in two states: GTPbound (active) and GDPbound (inactive). Thus, exchange of GDP for GTP stimulates Ras activation and enables this protein to recruit one of its down­stream effectors, Raf. So how does TCR stimulation result in activation of Ras? One of the ways in which Ras activation can be achieved is through the activity of GEFs (guaninenucleotide exchange factors) that promote exchange of GDP for GTP on Ras. One such GEF, SOS (son of sevenless), is recruited to phosphorylated LAT via the phosphotyrosinebinding protein GRB2 (Figure 7.8). Thus, phosphorylation of LAT by ZAP70 leads directly to the recruitment of the GRB2/SOS complex to the plasma membrane where it can stimulate activation of Ras through promoting exchange of GDP for GTP.

Figure 7.10 The Ras–MAP kinase pathway. Regulation of Ras activity controls kinase amplification cascades. A number of cell surface receptors signal through Rasregulated pathways. Ras cycles between inactive Ras–GDP and active Ras–GTP, regulated by guanine nucleotide exchange factors (GEFs) that promote the conversion of Ras–GDP to Ras–GTP, and by GTPaseactivating proteins (GAPs) that increase the intrinsic GTPase activity of Ras. Upon ligand binding to receptor, receptor tyrosine kinases recruit adaptor proteins (e.g., Grb2) and GEF proteins, such as Sos (“son of sevenless”), to the plasma membrane. These events generate Ras–GTP, which can now recruit the Raf kinase (also known as mitogen activated protein kinase, MAPK) to the plasma membrane, where it becomes activated by another membraneassociated kinase. Activation of Raf then leads to a cascade of further kinase activation events downstream, culminating in the activation of a battery of transcription factors, including Elk1. The Ras–MAPK cascade is frequently invoked by growth factors and other stimuli that trigger proliferation.

In its GTPbound state, Ras can recruit a kinase, Raf (also called MAPKKK, mitogenassociated protein kinase kinase kinase!), to the plasma membrane that then sets in motion a series of further kinase activation events culminating in phosphorylation of the transcription factor Elk1, in addition to many other transcription factors. Elk1 phosphorylation permits translocation of this protein to the nucleus and results in the expression of Fos, yet another transcription factor. The appearance of Fos results in the formation of heterodimers with Jun to form the AP1 complex that has binding sites on the IL2 promoter as well as on many other genes (Figure 7.11). Deletion of AP1 binding sites from the IL2 promoter abrogates 90% of IL2 enhancer activity.


Figure 7.11 Overview of TCRbased signaling. Signals through the MHC–antigen complex (signal 1) and B7 molecules (signal 2) initiate a cascade of protein kinase activation events and a rise in intracellular calcium, thereby activating transcription factors that control entry in the cell cycle from G0 and regulate the expression of IL2 and many other cytokines. Stable recruitment of CD4 or CD8 to the TCR complex initiates the signal transduction cascade through phosphorylation of the tandemly arranged ITAM motifs within the CD3 ζ chains, which creates binding sites for the ZAP70 kinase. Subsequent events are marshaled through ZAP70mediated phosphorylation of LAT; recruitment of several signaling complexes to LAT results in triggering of the Ras–MAPK and PLCγ1 signaling pathways. The latter pathways culminate in activation of a range of transcription factors including NFkB, NFAT, and Fos/Jun heterodimers. Note that other molecules can also contribute to this pathway but have been omitted for clarity. See main body of text for further details. DAG, diacylglycerol; ERK, extracellular signal regulated kinase; IP3, inositol trisphosphate; LAT, linker for activated Tcells; NFkB, nuclear factor kB; NFAT, nuclear factor of activated Tcells; OCT1, octamerbinding factor; Pak1, p21activated kinase; PIP2, phosphatidylinositol diphosphate; PKC, protein kinase C; PLC, phospholipase C; SH2, Srchomology domain 2; SLAP, SLP76 associated phosphoprotein; SLP76, SH2domain containing leukocytespecific 76 kDa phosphoprotein; ZAP70, ζ chainassociated protein kinase.

The phosphatidylinositol pathway

Phosphorylation of LAT by ZAP70 not only promotes docking of the GRB2/SOS complex on LAT, but also stimulates recruitment of the γ1 isoform of phospholipase C (PLCγ1) (Figure 7.8b). PLCγ1 plays a crucial role in propagating the cascade further. Phosphorylation of PLCγ1 activates this lipase thereby enabling it to hydrolyze the membrane phospholipid phosphatidylinositol bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3) (Figure 7.11). Interaction of IP3 with specific receptors in the endoplasmic reticulum triggers the release of Ca2+  into the cytosol that also triggers an influx of extracellular calcium (Figure 7.12). The raised Ca2+ concentration within the Tcell has at least two consequences. First, it synergizes with DAG to activate protein kinase C (PKC); second, it acts together with calmodulin to increase the activity of calcineurin, a protein phosphatase that can promote activation of an important transcription factor (NFAT) required for IL2 production.

Figure 7.12 An activated Tcell undergoes calcium flux. A Tcell receives a calcium signal (yellow glow) upon cognate interaction with a naive Bcell.

The Ca2+dependent activation of PKC by DAG is instrumental in the activation of yet another transcription factor, NFκB. NFκB factors that are involved in the regulation of transcription of many genes, including cytokines (such as IL2), as well as genes that can promote cell survival by blocking signals that promote apoptosis.

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