Biochem Chpt 12.3: Receptor Tyrosine Kinases

Question 1

How do receptor tyrosine kinases (RTKs) transduce extracellular signals by a mechanism fundamentally different from that of GPCRs?

Answer 1

RTKs have a ligand-binding domain on the extracellular face of the plasma membrane and an enzyme active site on the cytoplasmic face, connected by a single transmembrane segment. The cytoplasmic domain is a protein kinase that phosphorylates Tyr residues in specific target proteins—a Tyr kinase.

Question 2

What receptors are prototypes for RTKs?

Answer 2

The receptors for insulin and epidermal growth factor are prototypes for this group.

Question 3

What are the functions and effects of insulin?

Answer 3

Insulin regulates both metabolic enzymes and gene expression. Insulin does not enter cells, but initiates a signal that travels a branched pathway from the plasma membrane receptor to insulin-sensitive enzymes in the cytosol and to the nucleus, where it stimulates the transcription of specific genes

Question 4

Describe the structure of the insulin receptor

Answer 4

The active insulin receptor protein (INSR) consists of two identical alpha subunits protruding from the outer face of the plasma membrane and two transmembrane beta subunits with their carboxyl termini protruding into the cytosol—a dimer of alpha-beta monomers

Question 5

What is the functional relevance of the two structures consisting the insulin receptor?

Answer 5

The alpha subunits contain the insulin-binding domain, and the intracellular domains of the beta subunits contain the protein kinase activity that transfers a phosphoryl group from ATP to the hydroxyl group of Tyr residues in specific target proteins

Signaling through INSR begins when the binding of one insulin molecule between the two subunits of the dimer activates the Tyr kinase activity, and each alpha subunit phosphorylates three critical Tyr residues near the carboxyl terminus of the other beta subunit.

Question 6

What does this autophosphorylation in the insulin receptor achieve?

Answer 6

This autophosphorylation opens up the active site so that the enzyme can phosphorylate Tyr residues of other target proteins. A region of the cytoplasmic domain (an autoinhibitory sequence) that usually occludes the active site moves out of the active site after being phosphorylated, opening up the site for the binding of target proteins

Question 7

Describe an example of this insulin signalling naming a target

Answer 7

When INSR is autophosphorylated, one of its targets is insulin receptor substrate-1 (IRS-1). Once phosphorylated on several of its Tyr residues, IRS-1 becomes the point of nucleation for a complex of proteins that carry the message from the insulin receptor to end targets in the cytosol and nucleus, through a long series of intermediate proteins.

Question 8

Describe this long signalling of intermediary proteins in IRS-1 signalling. What overall effect does this have on the cell?

Answer 8

First, a P–Tyr residue of IRS-1 binds to the SH2 domain of the protein Grb2.

Several signaling proteins contain SH2 domains, all of which bind P–Tyr residues in a protein partner. Grb2 is an adaptor protein, with no intrinsic enzymatic activity. Its function is to bring together two proteins that must interact to enable signal transduction.

In addition to its SH2 domain, Grb2 also contains a second protein-binding domain, SH3, that binds to a proline-rich region of Sos, recruiting Sos to the growing receptor complex.

When bound to Grb2, Sos acts as a guanosine nucleotide–exchange factor (GEF), catalysing the replacement of bound GDP with GTP on Ras, a G protein.

When GTP binds, Ras can activate a protein kinase, Raf-1, the first of three protein kinases—Raf-1, MEK, and ERK—that form a cascade in which each kinase activates the next by phosphorylation. The protein kinases MEK and ERK are activated by phosphorylation of both a Thr and a Tyr residue.

When activated, ERK mediates some of the biological effects of insulin by entering the nucleus and phosphorylating transcription factors, such as Elk1, that modulate the transcription of about 100 insulin-regulated genes, some of which encode proteins essential for cell division. Thus, insulin acts as a growth factor.

Question 9

Thus describe this insulin-IRS 1 pathway in 7 steps

Answer 9

1. Insulin receptor binds insulin and undergoes autophosphorylation on its carboxyl-terminal Tyr residues.
2. Insulin receptor phosphorylates IRS-1 on its Tyr residues.
3. SH2 domain of Grb2 binds to P –Tyr of IRS-1. Sos binds to Grb2, then to Ras, causing GDP release and GTP binding to Ras.
4. Activated Ras binds and activates Raf-1.
5. Raf-1 phosphorylates MEK on two Ser residues, activating it. MEK phosphorylates ERK on a Thr and a Tyr residue, activating it.
6. ERK moves into the nucleus and phosphorylates nuclear transcription factors such as Elk1, activating them.
7. Phosphorylated Elk1 joins SRF to stimulate the transcription and translation of a set of genes needed for cell division.

Question 10

The proteins Raf-1, MEK, and ERK are members of three larger families, for which several nomenclatures are employed. To which families do each belong?

Answer 10

ERK is in the MAPK family (mitogen-activated protein kinases; mitogens are extracellular signals that induce mitosis and cell division).

Soon after discovery of the first MAPK enzyme, that enzyme was found to be activated by another protein kinase, which was named MAP kinase kinase (MAPKK; MEK belongs to this family).

When a third kinase that activated MAP kinase kinase was discovered, it was given the slightly ludicrous family name MAP kinase kinase kinase (MAPKKK; Raf-1 is in this family).

Question 11

What commonalities are shared within the families in regards to their phosphorylation?

Answer 11

Kinases in the MAPK and MAPKKK families are specific for Ser or Thr residues, and MAPKKs (here, MEK) phosphorylate both a Ser and a Tyr residue in their substrate, a MAPK (here, ERK).

Question 12

How is this insulin pathway reflective of a more general physiological scheme?

Answer 12

Biochemists now recognize this insulin pathway as but one instance of a more general scheme in which hormone signals, via similar pathways, result in phosphorylation of target enzymes by protein kinases. The target of phosphorylation is often another protein kinase, which then phosphorylates a third protein kinase, and so on. The result is a cascade of reactions that amplifies the initial signal by many orders of magnitude

Question 13

Name two other signals mediated by MAPK cascades

Answer 13

MAPK cascades mediate signaling initiated by a variety of growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF).

Question 14

What is another general scheme exemplified by the insulin receptor?

Answer 14

Another general scheme exemplified by the insulin receptor pathway is the use of nonenzymatic adaptor proteins to bring together the components of a branched signaling pathway

Question 15

How does the signalling pathway from insulin branch at IRS-1?

Answer 15

Grb2 is not the only protein that associates with phosphorylated IRS-1. The enzyme phosphoinositide 3-kinase (PI3K) binds IRS-1 through PI3K’s SH2 domain

Question 16

What happens once PI3K binds IRS-1?

Answer 16

Thus activated, PI3K converts the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3).

Question 17

What is the consequence of this conversion of PIP2 to PIP3?

Answer 17

The multiply charged head group of PIP3, protruding on the cytoplasmic side of the plasma membrane, is the starting point for a second signaling branch involving another cascade of protein kinases.

Question 18

Describe this second signalling branch initiated by PIP3 in the context of glycogen synthesis

Answer 18

When bound to PIP3, protein kinase B (PKB; also called Akt) is phosphorylated and activated by yet another protein kinase, PDK1.

The activated PKB then phosphorylates Ser or Thr residues in its target proteins, one of which is glycogen synthase kinase 3 (GSK3).

In its active, nonphosphorylated form, GSK3 phosphorylates glycogen synthase, inactivating it and thereby contributing to the slowing of glycogen synthesis. (This mechanism is only part of the explanation for the effects of insulin on glycogen metabolism.)

When phosphorylated by PKB, GSK3 is inactivated. By thus preventing inactivation of glycogen synthase in liver and muscle, the cascade of protein phosphorylations initiated by insulin stimulates glycogen synthesis

Question 19

How else may this IRS-1-PIP3 stemming pathway influence glucose metabolism?

Answer 19

In a third signalling branch in muscle and fat tissue, PKB triggers the clathrin-aided movement of glucose transporters (GLUT4) from internal vesicles to the plasma membrane, stimulating glucose uptake from the blood

Question 20

As in all signaling pathways, there is a mechanism for terminating the activity of the PI3K–PKB pathway. Describe this

Answer 20

A PIP3-specific phosphatase (PTEN in humans) removes the phosphoryl group at the 3 position of PIP3 to produce PIP2, which no longer serves as a binding site for PKB, and the signaling chain is broken.

Question 21

What is the clinical relevance of this termination mechanism of PIP3?

Answer 21

In various types of cancer, it is often found that the PTEN gene has undergone mutation, resulting in a defective regulatory circuit and abnormally high levels of PIP3 and of PKB activity. The result seems to be a continuing signal for cell division and thus tumor growth.

Question 22

The insulin receptor is the prototype for several receptor enzymes with a similar structure and RTK activity, but how similar are receptors for other growth factors?

Answer 22

The receptors for EGF and PDGF, for example, have structural and sequence similarities to INSR, and both have a protein Tyr kinase activity that phosphorylates IRS-1. Many of these receptors dimerize after binding ligand; INSR is the exception, as it is already an (aB)2 dimer before insulin binds.

[Stopped here pg 457 as rest not covered in lecture]