Regulating Target Protein Function

Question 1

Altering subcellular localisation:

Answer 1

• one way this can happen is by phosphorylation causing the binding of 14-3-3 proteins
• 14-3-3 proteins are a small family of proteins that usually exist as homodimers
• they bind phosphopeptides with the sequence R-S-X-pS-X-P (or variants of this motif)
• the symmetrical structure of 14-3-3 dimers creates a “horse-shoe” shape that bind two phosphorylation site sequences in two symmetrical pockets
• the phosphate groups (negative) bind clusters of basic, positively charged side chains on the 14-3-3 dimer:

Question 2

CONSEQUENCES OF 14-3-3 BINDING: 2 examples

Answer 2

• the two phosphorylated peptides are usually present on a single target protein
• binding of two phosphorylation sites on one protein by 14-3-3s increases the binding affinity

Example 1
- protein kinase PKB/Akt phosphorylates the transcription factor FoxO1 on multiple sites

• this causes binding of FoxO1 to 14-3-3 proteins, masking the nuclear localization sequence on FoxO1 but leaving its nuclear export sequence exposed
• since FoxO1 normally shuttles between the nucleus and the cytoplasm, activation of PKB/Akt will causes it to become trapped in the cytoplasm
• this explains how insulin, released in response to high blood glucose and which activates PKB/Akt, switches off expression of gluconeogenic genes, and hence liver glucose production

Example 2

• another example is the effect of AMPK on the Rab-GAP protein TBC1D1, which binds to intracellular storage vesicles containing the glucose transporter GLUT4 in muscle (Lecture 18)
• AMPK is activated by ATP depletion during contraction; it phosphorylates one site on TBC1D1 while other kinases phosphorylate a second site
• this causes 14-3-3 binding and dissociation of TBC1D1 from the GLUT4 vesicles, preventing its Rab-GAP activity from maintaining Rab proteins in their inactive GDP-bound state
• Rabs are thus converted to their active GTP state, promoting fusion of the storage vesicles with the plasma membrane; thus glucose uptake increases to fuel muscle contraction:

Question 3

PROMOTING DEGRADATION OF THE PROTEIN:

Answer 3

• phosphorylation may in some cases promote degradation of the protein, but we will discuss an example involving a different covalent modification, hydroxylation of the amino acid proline
• hypoxia-inducible factor-1a (HIF1a) promotes transcription of genes required during hypoxia, such as genes encoding the glycolytic pathway, which generates ATP even in absence of O2
• in normal [O2] (normoxia), HIF-1a is hydroxylated on proline, a reaction that requires oxygen:
• this triggers binding of HIF1a to VHL, a protein that has a binding pocket for hydroxylated HIF1a:
• VHL is an E3 ubiquitin ligase; VHL binding thus triggers polyubiquitylation of HIF1a and its consequent degradation by the proteasome
• thus, HIF1a is degraded in normoxia, but is stabilized in hypoxia due to lack of O2 for hydroxylation, promoting the transcription of genes such as glycolytic enzymes
• VHL is a tumour supressor

Question 4

FORMATION OF MULTIPROTEIN COMPLEXES:

Answer 4

• in some cases, protein phosphorylation creates binding sites for adapter domains
• e.g. SH2 domains recognize phosphotyrosine residues, with some specificity also for the amino acid three residues C-terminal to the P-Tyr (P+3 position)

Question 5

REGULATION OF ENZYME ACTIVITY:

Answer 5

• with some enzymes that are kinase targets, phosphorylation occurs at or near active site
• however in most cases phosphorylation sites are remote from the active site, so regulation is indirect – a good example of this is provided by the enzyme glycogen phosphorylase

Question 6

THE SYMMETRY MODEL FOR ALLOSTERIC REGULATION:

Answer 6

The model has the following key postulates:
- the protein must be a homo-oligomer, with two or more identical copies of each subunit (i.e. more than one identical subunit)

• within each oligomer, all subunits always have the same conformation (the so-called symmetry condition)
• the protein exists in two conformational states, the active R state and inactive T state
• equilibrium constant for the transition (allosteric constant or L) = [T]/[R] at equilibrium
• substrates preferentially bind to and stabilize R state, displacing equilibrium to the left
• activators also bind R state (at different site from substrate), displacing equilibrium to left
• conversely, inhibitors preferentially bind the T state, displacing the equilibrium to right
• thus, activators will increase the affinity for substrate, while inhibitors will decrease it
• Glycogen Phosphorylase is allosterically activated by AMP, and activated by phosphorylation from phosphorylase kinase
• Therefore glycogen phosphorylase has 4 states: (R, T, R-P, T-P)
• AMP has a large effect on the activity of R and T forms (dephosphorylated forms)

Question 7

STRUCTURE OF GLYCOGEN PHOSPHORYLASE:

Answer 7

• phosphorylase is a dimer
• when phosphorylated, the N-terminus flips, forming new interactions at the dimer interface
• note that the AMP-binding site and the Ser-P site are located at the dimer interface, far away from the active site where the cofactor, pyridoxal phosphate, is located
• changes at the dimer interface are transmitted to the active site by the alpha7 helix

Question 8

STABILIZATION OF R STATE BY AMP/PHOSPHORYLATION:

Answer 8

• structures of the dimers revealed a 10 degree rotation of the two subunits with respect to each other in the R state compared to the T state:
• in the T state, the alpha7 helices affect the active sites, restricting access to substrate
• in the R state, the alpha7 helices shift, allowing the substrate (glycogen) into the active site
• the binding sites for AMP and serine-P are located at the dimer interface
• binding of AMP/Ser-P stabilizes R state by “pulling” the subunits together on right-hand side
• thus, binding of AMP, or phosphorylation of Ser-14, promotes the active “R” conformation