Signaling proteins - Monomeric GTPases • Protein kinases • Adaptor proteins • Calcium-binding proteins Flashcards
How are GTPases inactivated? (in your answer, include the regulatory proteins that are
responsible for inactivating GTPases)
GTPases are inactivated by hydrolysing the GTP attached to them to GDP and phosphate. This process
(GTPase inactivation) is stimulated by GAPs (GTPase activating proteins)
Active GTPases are usually associated with cell membranes, while inactive GTPases occur in the
cytoplasm. Why is this the case?
The binding of GTP to GTPases (i.e. GTPase activation) causes the GTPase to change conformation and expose
either an amphipathic alpha-helix or lipid anchor that allows them to attach to lipid membranes.
What are the two main classes of protein kinases? Name three protein kinases you have
encountered in this course.
Ser/Thr and Tyr kinases.
REF,MEK, AND ERK
Though there are exceptions, protein kinases usually activate other proteins. How are these
activated proteins subsequently deactivated?
Protein kinases activate substrate proteins by attaching a phosphate group to them. The substrate proteins
are deactivated by phosphoprotein phosphatases that remove the phosphate.
An increase in cytoplasmic Ca2+ concentration often acts as a means to trigger rapid cell responses
during signal transduction.
i) Which two proteins are predominantly responsible for keeping the cytoplasmic Ca2+ concentration
below that of the external environment and ER?
ii) Which protein is responsible for the rapid entry of Ca2+ into cells from the external environment,
and under what conditions does it usually allow the entry of Ca2+?
iii) Based on material you have covered in the course, provide two examples of cell responses that
are triggered by an increase of cytoplasmic Ca2+ (you don’t have to describe the responses in detail,
just state what they are)
) i) SERCA and the plasma membrane Ca2+ ATPase.
ii) L-type Ca2+ channel. It is voltage-gated, thus allows entry of Ca2+ when the plasma membrane depolarises.
iii) Possibilities are: Muscle contraction; a fusion of synaptic vesicles to membranes (or release of
neurotransmitters); release of hormones by gland cells.
Describe how activation of receptor tyrosine kinases by two ligands cause the binding of adaptor
proteins to their cytoplasmic domains.
The two ligands bind the separate RTKs. This causes the RTKs to dimerise (bind to each other). This activates
the Tyr kinase activity of their cytoplasmic domains, and they phosphorylate Tyr residues on each other’s
cytoplasmic domains. The phosphorylated tyrosines are recognised and bound by adaptor proteins.
i) Which signaling pathway is activated by growth factors and responsible for stimulating cell
proliferations?
ii) Which protein acts as a guanine nucleotide exchange factor (GEF) in the pathway?
iii) Describe the reactions in the pathway after activation of Raf (MAP kinase kinase kinase).
i) MAP Kinase pathway.
ii) Sos (it stimulates exchange of GDP for GTP by Ras GTPase)
iii) Raf (or MAP kinsase kinase kinase) phosphorylates and activates MEK (or MAP kinase kinase). MEK in turn
phosphorylates and activates ERK (or MAP kinase). ERK moves into the nucleus and phosphorylates
transcription factors. The transcription factors stimulate the transcription of genes involved in cell
proliferation
Describe how protein kinase A (PKA) is activated by G-protein coupled receptors (GPCRs), and
give an example of a pathway that uses this mechanism (just briefly state the pathway and its
function – you don’t have to describe it is detail).
Binding of a ligand to a GPCR causes the cytoplasmic domain of the GPCR to bind to a G protein and
stimulate GDP/GTP exchange by the alpha subunit of the G protein (Gα). When attached to GTP, Gα dissociates
from the other subunits of the G protein and binds to an effector protein – adenylyl cyclase in this case. This
activates adenylyl cyclase to convert ATP to cAMP. The cAMP binds to and activates PKA. (if you really wanted
to be complete, you could add that binding to adenylyl cyclase stimulates Gα to hydrolyse GTP to GDP and
phosphate. This deactivates it – it detaches from the effector protein and reattaches to the beta and gamma
subunits of the original G protein).
Examples of pathways:
Glucagon signaling, which triggers breakdown of glycogen to glucose in muscle and liver cells
Adrenalin signaling, which increases the rate and strength of heart muscle contraction (or stimulates smooth
muscle cell relaxation).
How does activation of a GPCR effector protein lead to an increase in cytoplasmic Ca2+
concentration?
Phospholipase C is a GPCR effector protein which cleaves PIP2 to form DAG and IP3. The IP3 binds to an IP3
receptor on the membrane of the ER. The IP3 receptor is a ligand gated Ca2+ channel, thus binding of IP3
causes it to open and allow the release of Ca2+ from the ER into the cytoplasm.
Describe how steroid hormones trigger gene transcription in target cells
The hormone diffuses across the plasma membrane and binds to a steroid hormone receptor in the
cytoplasm of the target cell. The receptor dimerises, causing it to expose a nuclear localisation signal and move
into the nucleus. There the receptor acts as a transcription factor – it binds to steroid hormone response
elements (DNA sequences) in the promoters of genes, thus stimulating their transcription.
How does the substrate protein activated by Protein kinases deactivate?
by removal of the phosphate group by a specific phosphoprotein
phosphatase.
Signaling pathways thus often consist of a series of protein kinases that sequentially
phosphorylate and activate each other-they form what?
Signaling cascades
What is another name for docking proteins?
Adaptor proteins
What is the function of Adaptor proteins
Adaptor proteins often bind to signaling receptors occupied by ligands
(signaling molecules), thus recruiting additional signaling proteins to the activated receptor
What are the domains that enable the binding of adaptor molecule to the protein receptors ?
SH2 /SH3 and PTB
