Intro to Pharmacology and Toxicology Topics   

Regulation and Substrate Recognition

Kinases and phosphatases are regulated by ligands, by controlling their amount or location and by phosphorylation..

PKA, PKC and PP2B (calcineurin) are an examples of ligand-binding regulation. PKAs have a regulatory domain that acts as a pseudosubstrate, known as the hinge region, that mimics phosphorylation sites in the substrate, like RI(RRXA) or RII(RRXS).

PKC and the beta-adrenergic receptor kinase (beta-ARK) are examples of kinases regulated by controlling the amount or localization of protein. Beta-ARK is responsible for the desensitization of the beta-adrenergic receptor, by phosphorylation of the receptor. This allows arrestin to bind to the receptor and prevent interaction with Gs.

MAP kinase/ERK2 is regulated by phosphorylation. It has a Thr and a Tyr which can be phosphorylated, changing the conformation of the active site (activation). Other phosphoproteins activated by phosphorylation include CAPK, cdk2, Src and InsR..

Cyclin-dependent protein kinases (cdk2) are regulated by binding cyclin and by phosphorylation Human cdk2 is activated by binding to cyclin and phosphorylation at Thr160, whereas phosphorylation at either Tyr15 or Thr14 or both inactivates the cyclin-cdc2 complex. Wee1 is the tyrosine kinase that catalyzes the phosphorylation at Tyr15. Cdc25 is a dual specificity phosphatase that counteracts the action of wee1 by phosphorylating Tyr15 and Thr14. Cdc25 is activated by phosphorylation by GSK-3 followed by a conformational change (to the active form) and autodephosphorylation to keep the active conformation.

There are multiple levels of specificity determinants for both kinases and phosphatases. Specificity may depend on the geometry of the active site, its amino acid sequence and/or targeting and docking interactions.

The geometry of the active site determines selectivity for Tyr, Ser, Thr or their phosphates. The amino acid sequence surrounding the active site is an important determinant of specificity for kinases, as many kinases have a preferred sequence:

PKA:                           R-R-X-(S/T)-F

Calmodulin kinase:   R-X-X-(S/T)

MAPK:                        P-X-S-(S/T)-P

CDC2:                        S-P-X-(K/R)

Targeting and docking interactions also determine selectivity. Substrates may bind to the catalytic subunit or to other part of the enzyme, including regualatory subunits.

Continue to "Cell Excitability " or take a quiz: [Q1] [Q2] [Q3] [Q4].

Need more practice? Answer the review questions below (after sponsor).

1- List 3 ways of controlling kinases and phosphatases.

2- List 3 examples of ligand-binding regulation in phosphoproteins.

3- Why do the pseudosubstrate in PKA bind to the catalytic site?

4- List 2 examples of phosphoproteins regulated by amount/localization.

5- What is beta-ARK?

6- List examples of phosphoproteins regulated by phosphorylation.

7- What is the effect of phosphorylation on MAPK?

8- How is cdc2 regulated?

9- How is the cyclin-cdc2 complex deactivated?

10- What is the activity of cdc25?

11- How is cdc25 activated?

12- List 3 determinants of kinase/phosphatase specificity.

13- What is determined by the geometry of the active site of phosphoproteins?

14- List 4 kinases and their preffered binding sites.

15- Where do substrates bind in phosphoproteins?

Continue scrolling to answers below (after sponsor).

Hey! DON'T PEEK!!! Finish the questions fist!

1- List 3 ways of controlling kinases and phosphatases.
ligands
controlling amount and localization
phosphorilation

2- List 3 examples of ligand-binding regulation in phosphoproteins.
PKA
PKC
PP2B (calcineurin)

3- Why do the pseudosubstrate in PKA bind to the catalytic site?
Mimics phosphorylation sites in the substrate

4- List 2 examples of phosphoproteins regulated by amount/localization.
PKC
beta-ARK

5- What is beta-ARK?
Enzyme responsible for desensitization of beta-adrenergic receptors by phosphorylating the receptor. Allows arrestin to bind to the receptor and prevent interaction with Gs.

6- List examples of phosphoproteins regulated by phosphorylation.
MAPK/ERK2
cAPK
CDK2
Src
InsR

7- What is the effect of phosphorylation on MAPK?
It is activated by phosphorylation of Thr and Tyr.

8- How is cdc2 regulated?
By binding to cyclin and phosphorylation at Thr160

9- How is the cyclin-cdc2 complex deactivated?
By phosphorylation of either Tyr15 or Thr14 or both by wee1.

10- What is the activity of cdc25?
A dual specificity phosphatase that counteracts the action of wee1 by dephosphorylating Tyr15 and Thr14.

11- How is cdc25 activated?
By phosphorylation by GSK-3 followed by a conformational change to the active form and autodephosphorylation, keeping the active conformation.

12- List 3 determinants of kinase/phosphatase specificity.
geometry of the active site
amino acid sequence
targeting and docking interactions

13- What is determined by the geometry of the active site of phosphoproteins?
Selectivity for either Tyr, Ser, Thr or their phosphates.

14- List 4 kinases and their preffered binding sites.
PKA: RRX(S/T)F
Calmodulin kinase: RXX(S/T)
MAPK: PXS(S/T)P
cdc2: SPX(K/R)

15- Where do substrates bind in phosphoproteins?
May bind to the catalytic subunit or other parts of the enzyme, including regulatory subunit.

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