These are notes from lecture 10 of Harvard Extension’s Cell Biology course.
Lecture 9 introduced signal transduction. In a subset of signal transduction pathways, ligand binding to a receptor causes phosphorylation of cytosolic proteins, changing their ability to regulate gene transcription. Today’s lecture will provide several examples of these sorts of pathways.
Transforming growth factor beta (TGF-beta or TGF-β) is a secreted protein that acts as a first messenger in many signaling pathways, with an emphasis on cell proliferation, differentiation, and immune responses. Actually, TGF-β refers to any of three different secreted proteins named TGF-β1, 2, and 3 and coded-for by genes TGFB1, 2, and 3. TGF-β is a cytokine, a term which appears to have no rigorous definition but which loosely refers to signaling molecules involved in immune responses. To make things more confusing: here I’m discussing TGF-β specifically, but TGF-β is also the name of a whole superfamily of proteins which includes most of the bone morphogenetic proteins (BMPs).
The full TGF-β genes each have ~400 codons, of which ~20 are a signal peptide and ~270 are a latency associated peptide (LAP) which keeps the remaining ~110 amino acids inactive. Most cells produce and secrete the full (inactive) form of the protein all the time. Regulation is post-translational: when a TGF-β signal is desired, the LAP is cleaved off and then pairs of the ~110 amino acid active TGF-β peptides dimerize (binding covalently via an intermolecular disulfide bond) to form a functional signaling molecule.
Once that mature signaling molecule is formed, it can bind to – you guessed it – TGF-β receptors. There are three such receptors, called RI, RII and RII and coded-for by genes TGFBR1, 2 and 3. They are single-pass transmembrane proteins. All of them are serine/threonine kinases (STKs), meaning they can phosphorylate the -OH group in S and T amino acids on other proteins. In general STKs are quite promiscuous: though they must recognize a motif in order to phosphorylate their substrates, they are not specific to a single protein and usually can phosphorylate a whole class of proteins that share the relevant motif.
In the case of the TGF-β receptors, we’ll discuss a particular apoptotic signaling pathway in which the substrates are the SMAD proteins, of which there are several. They are broken into three subgroups:
- Receptor-regulated SMADs (R-SMADs)
- The common mediator SMAD (co-SMAD, aka SMAD4)
- Inhibitory SMADs (I-SMADs).
There are several different exact pathways by which signaling can occur. For simplicity, here is one of them. A TGF-β dimer binds an RII receptor, causing it to phosphorylate itself and recruit an RI receptor. RI then phosphorylates SMAD3, causing a conformational change that exposes both its nuclear localization sequence (NLS) and its co-SMAD binding site. SMAD3 then binds SMAD4, importin grabs the NLS and drags the whole complex into the nucleus, where SMAD3 and SMAD4 each bind to (separate) sites in the promoter region of a target gene, activating transcription. However, there is also a counter-regulatory pathway wherein Ski protein (gene: SKI) will bind the SMAD3/SMAD4 complex at the promoter and recruit histone deacetylases (HDACs) to epigenetically repress gene expression.
Here’s a video introducing the pathway generally:
And here is a super brief rundown of one example of a JAK/STAT pathway. Erythropoietin (Epo; gene: EPO) is a peptide signaling molecule secreted when additional red blood cells (erythrocytes) are needed, such as when you go to a high altitude – in fact, it’s used for blood doping. Epo binds to a homodimer of two Epo receptor proteins (EpoR; gene: EPOR) which are transmembrane proteins each bound to a JAK2 protein on the cytosolic side. When bound by Epo, the EpoRs undergo a conformational change that brings the two JAK2s into a position where they phosphorylate each other, thus activating each other. JAKs are tyrosine kinases, and once active they phosphorylate several Y amino acids on the EpoRs. Those phosphotyrosines act as ‘docking points’ for STAT proteins which have SH2 (Src homology 2) or PTB (phosphotyrosine-binding) domains. Once ‘docked’ the STATs get phosphorylated by JAK2, whereupon they dimerize and, thus activated, move into the nucleus to begin binding DNA and regulating transcription. In the specific case of the Epo pathway, the relevant STAT is STAT5 (actually STAT5A & B), which acts by blocking apoptosis of red blood cell progenitors. In bone marrow, such progenitors are constantly developing and then apoptosing; by blocking that apoptosis, STAT5 promotes proliferation of red blood cells.
Two types of proteins are involved in downregulation of JAK/STAT pathways: SHP1 and SOCS. SHP1 (gene: PTPN6) binds to the receptor and dephosphorylates the JAK, deactivating it and causing a short-term reduction in signal strength. SOCS proteins mediate more long-term regulation through several mechanisms. They also have SH2 domains that bind to the activated receptor, occupying those sites and preventing other proteins (like STATs) from being able to bind there. SOCS1 can also bind to JAK2 itself, preventing its catalytic activity. SOCS can also recruit E3 ubiquitin ligases to JAK2, causing the latter to be degraded. And SOCS2 can also downregulate expression of the receptor gene.
receptor tyrosine kinase pathways
These pathways regulate cell proliferation, differentiation, survival and metabolism. The signaling molecules include FGF, NGF, EGF, PDGF. Each of these first messengers binds to a receptor tyrosine kinase (RTK). Here the receptor itself is a kinase (unlike in JAK/STAT above, where the receptor was complexed with a separate kinase). RTKs are single-pass Type III membrane proteins, with a C-terminal cytosolic domain that has kinase activity, a transmembrane domain, and an N-terminal extracellular domain that binds the signaling molecule. RTKs exist as monomers but dimerize when bound by the signaling molecule, causing them to phosphorylate each other. The phosphotyrosines on the receptors then become docking sites for SH2 domains of other proteins, which dock there and get phosphorylated.
A classic example of RTKs is the HER family of receptors. For instance, HER1 is the receptor for epidermal growth factor (EGF). HER2 is the target of Herceptin (trastuzumab), the famous antibody used in treatments for some breast cancers
RTK signaling is regulated in part through clathrin-mediated endocytosis leading to lysosomal degradation of the receptor. This makes it possible to stop the signal transduction cascade even when the ligand is still present. Following endocytosis, about 50% of the vesicles end up recycled to the cell surface and only 50% lead to degradation, so this does not completely stop the pathway.
Almost all RTKs can activate the Ras and MAP kinase pathways. Ras is a small monomeric G protein. When active it activates MEK which activates MAPK which then moves into the nucleus and phosphorylates transcription factors.
Here are two other very important signal transduction pathways that regulate gene transcription:
To see if a gene’s transcription is upregulated in response to a signal, introduce the signal to test cells and compare to control. Do qPCR on the transcripts of interest, controlling for amount of input RNA by normalizing to some housekeeping genes’ level.
Or instead of qPCR, you can introduce the gene of interest on a plasmid with a luciferase reporter gene, and measure the luminescence.