05/10/2016

Cell signalling

Richard Killick internal tel: 0529

richard.1.killick@kcl.ac.uk

Cell signalling 1

• What is cell signalling?

• Overview

• Some basics

• Selection of specific pathways

• Insulin signalling

• Notch signalling

• The Wingless/wnt pathway(s)

Cell signalling 2

…specific pathways continued

• p53

• Nuclear Hormone Receptors

• EGF - ErbB/neuregulin

• Pathology – Cell-cell signalling in disease

05/10/2016

What is cell signalling

Cell signalling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. Wikipedia

How information is passed between cells and how they respond.

Aim is to contrast and compare the different mechanisms that cells have developed to communicate with one another.

Discuss how dysregulation of cell signalling may underlie many diseases, particularly neurodegeneration.

A eukaryotic cell

05/10/2016

In a broad sense Cell Signalling does include Neurotransmission - the means by which neurons pass information.

There is overlap between Metabotropic neurotransmission and some of the cell-cell signalling pathways we will discuss.

But when we talk about Cell Signalling here we are not includingneurotransmission.N.B. When answering questions with Cell Signalling in the title trynot to dwell on neurotransmission.

The cell (or plasma) membrane

A lipid “bi-layer” with proteins attached,or passing through, it.

05/10/2016

Intracellular

Intracellular

Extracellular

Membrane

Membrane

Membrane bound receptor

Membrane bound ligand

Soluble ligand

The passing of information relies on

Receptors and their Ligands (mostly)

Reception

Transduction

Response

Time scale of response - can be from seconds to hours

LigandReceptor

Response

Signalling cascade

05/10/2016

Post translational modifications

PhosphorylationPhosphoprotein - A protein which can be phosphorylated – i.e. a phosphate group can be added to one or several specific amino acids.Kinase (or phosphorylase)Enzymes which catalyse the addition of a phosphate are called kinases.(kinase - an enzyme that catalyzes the transfer of a phosphate group from ATP to a specified molecule. ORIGIN early 20th cent.: from Greek kinein ‘to move’ + -ase).

This only occurs at Serine, Threonine or Tyrosine residues.

**

*

Post translational modifications

De-phosphorylation PhosphataseAn enzyme that catalyse the removal of a phosphate is a phosphatase.(phosphatase - an enzyme that catalyzes the hydrolysis of organic phosphates in a specified (acid or alkaline) environment.

There are many kinase families. Phosphatases can be subdivided based upon their substrate specificity giving five major families.

The addition or removal of a phosphate group may…

- Create a binding site for another protein.- Alter a binding site such that a ligand cannot bind.

- Stabilise a region of protein structure.- Destabilise a region of protein structure.

- Protect a protein from degradation.- Increase the likelihood of degradation.

05/10/2016

Ribosylation ADP ribose polymers performed by PARP

The response (in many cases) is transcriptional, a change in gene expression - the switching on or off of a certain “target” genes.

Gene expression, or transcription, is regulated by transcription factors

In molecular/cellular biology, a transcription factor (sometimes called a sequence-specific DNA binding factor) is a protein that binds to specific sequences of DNA and thereby controls the transcription of genetic information from DNA to RNA.

Transcription factors perform this function alone, or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme which performs the transcription of DNA into RNA) to specific genes.

Simplistically, TFs work by switching genes on or off!

05/10/2016

Transcription factors are modular in structure and often contain the following domains:

DBD - DNA-binding domain which attach to specific sequences of DNA (enhancer or promoter sequences) adjacent to regulated genes. DNA sequences which bind transcription factors are often referred to as response elements.

TAD - Trans-activating domain which contain binding sites for other proteins such as transcription co-regulators.

SSD - Signal sensing domain (optional e.g., a ligand binding domain) which senses external signals and in response transmits these signals to the rest of the transcription complex resulting in up or down regulation of gene expression. Alternatively the DBD and signal sensing domains may reside on separate proteins that associate within the transcription complex to regulate gene expression.

A defining feature of transcription factors is that they contain one or more DNA binding domains (DBDs), which attach to specific sequences of DNA adjacent to the genes that they regulate. Additional proteins such as co-activators, chromatin remodelers, histone acetylases/deacetylases, kinases, and methylases, while also playing crucial roles in gene regulation, lack DNA binding domains, and therefore are not true transcription factors.

DNA binding Transcription factors

The insulin pathway

Insulin is a small peptide hormone produced in the pancreas by the islets of Langerhans, which regulates the amount of glucose in the blood.

Lack of insulin production causes a form of diabetes referred to as Type I diabetes mellitus (DM).

Loss of sensitivity of a cell to insulin results in Type II DM.

ORIGIN from Latin insula ‘island’ + -in

Insulin signalling

05/10/2016

The insulin receptor

The insulin receptor (IR) is a dimer of two proteins. Each IR protein is cleaved into the alpha and beta subunits, which then bind each other through cysteine bonds. The mature receptor is thus composed of two extracellular alpha subunits and two transmembrane beta subunits linked together by disulphide bonds. Binding of insulin to the alpha subunit induces a conformational change resulting in the autophosphorylation of a number of tyrosine residues present in the beta subunit.

IR is a receptor tyrosine kinase

Insulin binding activates the insulin receptor (IR)

a tyrosine kinase receptor.

Tyrosine kinase receptors mediate their activity by causing the addition of a phosphate group to specific tyrosines on certain proteins within a cell.

Upon ligand (insulin) binding the IR undergoes a

conformational change leading to autophosphorylation.

The phosphorylated residues are then recognised by

phosphotyrosine-binding (PTB) domains of adaptor

proteins, in this case members of the insulin receptor

substrate family (IRS).

Binding of IRS to IR leads to the phosphorylation of key tyrosine residues on IRS proteins, which are recognised by the Src homology 2 (SH2) domain of the p85 regulatory subunit of PI 3-kinase (a lipid kinase).

The catalytic subunit of PI3-kinase, p110, then phosphorylates phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2) leading to the formation of PtdIns(3,4,5)P3.

A key downstream effector of Ptd(3,4,5)P3 is Akt (also known as PKB), which is recruited to the plasma membrane. Activation of AKT also requires the protein kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1), which in combination with an as yet unidentified kinase leads to the phosphorylation of AKT. Once active, AKT enters the cytoplasm where it leads to the phosphorylation and inactivation of glycogen synthase kinase 3 (GSK3). GSK3 phosphorylates and inhibits glycogen synthase, an enzyme that catalyses the final step in glycogen synthesis. Thus the inactivation of GSK3 by Akt promotes glucose storage as glycogen.

Insulin signalling

Glut4 containing vesicles are transported to the cell surface along fibres making up the cytoskeleton and are placed into the plasma membrane by docking proteins.

In addition to regulating glycogen synthesis insulin signalling also leads to the relocation of the glucose uptake molecule, GLUT4, into the cell membrane.

GLUT4 translocation

05/10/2016

Insulin signalling pathway

Insulin then causes the activation of PI3K which subsequently phosphorylates Akt. Akt then phosphorylates FOXO1, causing its nuclear exclusion. Phosphorylated FOXO1 is then ubiquitinated and degraded by the proteosome. The phosphorylation of FOXO1 is irreversible; this prolongs insulin's inhibitory effect on glucose metabolism and hepatic liver production. Transcription of glucose-6-phosphatase subsequently decreases, which consequently decreases the rates of gluconeogenesis and glycogenolysis.

Transcriptional effects of insulin are via the transcription factor FOXO1 (Forkhead [FKHR] Box O1).

The actions of insulin are countered by Glucagon - another small peptide hormone made in the pancreas. Made when glucose levels are low.

Glucagon opposes insulin

05/10/2016

Notchsignalling

Notch is derived from the “notched wing” phenotypeof the the fruit fly, Drosophila.

Notch wings, [left to right] average, extreme condition, nearly normal, T.H.Morgan, THE PHYSICAL BASIS OF HEREDITY, 1919.

The Notch receptor

The Notch locus encodes a large receptor protein with a single membrane spanning domain

05/10/2016

Notch signalling

05/10/2016

05/10/2016

What does Notch signalling do

One of the first to be discovered was its role in theknown as lateral inhibition

Many things!

05/10/2016

The Drosophila Notch gene is a “neurogenic locus”

The neurogenic loci comprise a small group of genes which are required for proper division between the neural and epidermal pathways of differentiation within the neuroectoderm. Loss of neurogenic gene function results in the misrouting of prospective epidermal cells into neuroblasts.

Loss of Notch leads to an excess of neurons.

Conversely the over expression of mammalian N1 in mouse brain leads to a small brain phenotype due to a reduction in neurons due to excessive apoptosis.

Wingless/wnt signaling

05/10/2016

The Wingless or (wnt) pathway

wnt is a contraction of Wingless and int (for integration)

In Drosophila -catenin is called armadillo

05/10/2016

The wnt--catenin or canonical wnt pathway

The wnt--catenin or canonical wnt pathway

In unstimulated cells, CKI phosphorylates -catenin on S45, priming it for subsequent phosphorylation by GSK-3 (S41, S37, S33), which targets beta-catenin for ubiquitylation and proteasomal degradation. The ankyrin repeat protein, Diversin (Div), may help recruit CKI to the destruction complex. Wnt stimulation activates the receptor Frizzled, which then signals through Dishevelled (Dvl), using an unclear mechanism, to inactivate beta-catenin phosphorylation. Unphosphorylated -catenin accumulates and then translocates to the nucleus where it transactivates genes regulated by TCF/LEF transcription factors. The GSK-3-binding protein (GBP/FRAT) may be involved in transmission of a Wnt signal by regulating binding of GSK-3 to the scaffold protein, axin.

GSK3

05/10/2016

The basic canonical wnt pathway

Cerberus and sFRPs soluble frizzleds - mop up wntsbefore they can bind receptor complex

05/10/2016

There are other secreted inhibitors - the Dickkopf family

These do not “mop up” wnts but separate co-receptor proteins

A Wnt Survival Guide: From Flies to Human Disease Andy J. Chien1, William H. Conrad1 and Randall T. Moon1

Canonical Ca2+ PCP

Text

SnapShot: Noncanonical Wnt Signaling PathwaysMikhail V. Semenov,1 Raymond Habas,2 Bryan T. MacDonald,1 and Xi He1

05/10/2016

Fig. 1. Dishevelled mediates at least three signaling cascades. (A) The canonical WNT pathway. WNT signals are received by a Frizzled (FZ) receptor and a LRP co-receptor. The signal is transduced through Dishevelled and various other proteins, leading eventually to the stabilization of b-Catenin, which then together with LEF/TCF controls the transcription of target genes. (B) The non-canonical or planar cell polarity (PCP) cascade. A signal is received by a FZ receptor (in Drosophila, no WNT has yet been implicated in PCP signaling in vertebrates, although WNT11 and WNT5a are necessary), and transduced via Dishevelled to RAC and RHO, which then activate downstream targets to modulate the actin cytoskeleton. (C) The WNT/Ca2+ pathway. WNT signaling through the FZ receptors and Dishevelled and G-proteins leads to the release of intracellular calcium and signaling via Phospholipase C, CamK2 and PKC (Miller et al., 1999a; Sheldahl et al., 1999; Sheldahl et al., 2003). Image reproduced with permission from BioMedCentral (see Habas and Dawid, 2005). APC, Adenomatous Polyposis Coli; CKI, Casein kinase 1; CamK2, calcium/calmodulin-dependent kinase 2; GBP, GSK3 Binding Protein; GSK3, Glycogen synthase kinase 3; JNK, Jun kinase; LRP, Low-density lipoprotein receptor-related protein; PKC, Protein kinase C; TCF/LEF, Lymphoid Enhancer-Binding Factor/T-Cell Specific Transcription Factor; b-TrCP, Beta-Transducin Repeat Containing Protein; ROCK, RHO-associated coiled-coil forming kinase

Ca2+ PCP

Core PCP

05/10/2016

The wing hairs of Drosophila are an excellent model to study PCP signaling. In wild type (left) all hairs point in the same direction towards the distal tip of the wing. In animals mutant for a gene controlling PCP, wing hair polarity is disturbed (right).

Planar Cell Polarity

What is the interest in an obscure pathway like Planar Cell Polarity for neuroscientists?