Non Receptor Tyrosine Kinases

Question 1

Tyrosine kinases can be divided into 2 main groups:

Answer 1

Receptor Tyrosine Kinases (RTKs)

Non receptor tyrosine kinases. (NRTKs)

Question 2

Non receptor tyrosine kinases:

Answer 2

10 sub families of non-receptor tyrosine kinases

7 subfamilies contain SH2 domains (Src, Abl, Btk, Fes, Syk, Csk and JAK families)

Tec, Frk and Fak families do not have SH2 domains

SH2 domains can regulate the activity of the kinase domain, but the mechanisms behind this vary depending on the family

Question 3

Src family kinases:

Answer 3

Cause Cell proliferation, Cell survival, Cell adhesion, Cell migration. Src family kinases are oncogenes.

Two key phosphorylation sites:

• Kinase domain: activation, autophosphorylation site
• C-terminal peptide: inhibitory, catalysed by CSK

Myristoylation of "unique domain"
SH3 domain
SH2 domain
Kinase Domain
C-Terminal peptide

Myristoylation: attachment of a myristol group from myristic acid via an amide bond to an N-terminal glycine residue. Allows for a weak interaction between the protein and a lipid membrane

Src family kinases can activate further non receptor tyrosine kinases

Question 4

FcEpsilon Receptor

Answer 4

Activation of mast cells occurs through this receptor
Tetramer
Binds IgE antibodies
alpha chain – binds IgE via Ig domains

beta chain –contain ITAM motif, binds the Src family tyrosine kinase Lyn

gamma chain – 2 copies bound by disulfide bridge, contains ITAMs that recruit Syk

Question 5

Stimulation of degranulation:

Answer 5

FcEpsilon Receptor is activated by clustering

SYK is the key kinase that allows activation of downstream signaling

PKC and Ca2+ signaling drive degranulation

MAPK (and NFkappaB) activation induce cytokine production

MAPKs (via PLA2) promote prostaglandin and leukotriene production

Question 6

Cytokines:

Answer 6

Key regulators of immunity
Cytokines mainly act to module the function of their target cells
Different cytokines have specific receptors – cytokines have been subdivided into families based on homology that the types of receptor they activate
Can act in autocrine or paracrine fashion
Some cytokines act to regulate immune cell development and hemostasis (eg IL-7) other act in pro-inflammatory (TNF, IL-6, IL-1) or anti-inflammatory (IL-10) responses
Some cytokines are monomeric (eg IL-1), others are dimers (eg IL-12) or trimers (eg TNF)

Question 7

JAK STAT signalling

Answer 7

JAK (Janus kinase)
Tyrosine kinase
4 isoforms: JAK1, JAK2, JAK3, TYK2

STAT (Signal Transducer and Activator of Transcription)
Transcription factor
7 isoforms: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6

JAK activates STATs. Very important in the immune system.

Activated by cytokine receptors or some receptor tyrosine kinases

Question 8

STAT function

Answer 8

STATs are transcription factors

Can act as homo- or heterodimers

While specificity exists between different STAT proteins, there is also a degree of functional redundancy

Two main response elements described

Most active GAS sites promote transcription (but can also inhibit or have no effect on transcription)

In both proximal and distal binding sites STAT may from complexes with other transcription factors

Question 9

Interferon signalling:

Answer 9

Three main groups of interferon, type I, type II and type III:

Type I: IFNalpha, IFNbeta
- produced by many cells types in response to viral
infection
- induces an “anti-viral” state in nearby cells
- can also modulate immune cell function

Type II: IFNgamma

• produced by some immune cells (NK cells and T cells)
• activates macrophages
• modulates B cell and T cell function

Question 10

STAT binding to GAS elements:

Answer 10

STATs can bind to GAS (Gamma interferon activation site) sequences

All STAT isoforms can bind GAS elements, although specific does exists at the level of individual promoters in cells

Original studies focused on identifying GAS elements associated with gene promoters

Next generation sequencing has highlighted additional roles for STATs

Question 11

IRF: Interferon Regulatory Factor

Answer 11

9 isoforms
Involved in regulating transcription in innate and adaptive immune cells
Most act as dimers (eg IRF3 downstream of TLR3 signalling)
IRF9 is unusual in that it binds to STAT2

Question 12

Negative regulation of JAK-STAT signalling:

Answer 12

SHP-1:
Protein tyrosine phosphatase
Recruited via its SH2 domains
Dephosphorylates receptor and/or JAK

PIAS (Protein Inhibitor of Activated STAT):
4 genes
Binds to STAT proteins and blocks their ability to bind DNA
Can act as an E3 ligase for Sumo (Sumo is a ubiquitin related protein)

SOCS (Suppressor of Cytokine Signalling):
8 genes (SOCS1 to 7 and CIS)
Have SH2 domain and SOCS box
SH2 binds to p-Tyr and blocks access to STATS
SOCS may recruit ubiquitination machinery and target JAKs for proteosomal degradation
SOCS 1 and 3 have a kinase inhibitory domain in their N terminus
Transcription of SOCS genes can be induced by JAK-STAT signalling
SOCS3 can determine signalling outcome

Question 13

NRTK overview:

Answer 13

e.g.

IFN -> receptor -> JAK activity -> Phosphorylation of STATs -> Effect (such as GAS elements) to promote transcription