Chemical Biology (Köhn)

Involved in Motility, growth, differentiation, death/survival pathways

Phosphorylation balance decides between health and disease

Aberrant Phosphorylation -> Oncoproteins -> uncontrolled growth & proliferation

• phosphatases evolved in distinct, separate families with different mechanisms -> phosphatases can be holoenzymes or single/multidomain proteins

• kinases evolved from the same ancestor, use ATP for catalysis -> are most commonly multidomain proteins

• differences in function & regulation

Protein Serine/Threonine Specific phosphatases: Metal-dependent protein phosphatases (PPM), Phosphoprotein Phosphatases (PPP)-like: Catalysis metal-ion dependent, holoenzymes (only catalytic subunit, interacts with many regulatory proteins)

• Bsp PPPLs: PP1, PP2A

Protein Tyrosine Phosphatases (PTPs) - cytosolic & Receptor-like PTPs -> classical PTPs: dephosphorylation at tyrosine, multidomain enzymes

Dual specificity phosphatases DUSPs: some dephosphorylate p-Ser/Thr, Small

• All PTPs Catalysis by active cysteine residue

• Bsp PTP: SHP2

• Protein tyrosine phosphatase (in Ras - Raf - Mek - Erk)

• Multidomain protein, PTP domain contains active site, dephosphorylates pTyr, SH2 domains recognize pTyr & bind it

• Proto-oncogene & needs to be inhibited in cancer to reduce growth signaling

• Active site inhibitors target the active site

• Allosteric site inhibitor targets a different site but also leads to inhibition, Bsp SHP099 - keeps SHP2 in closed conformation

• de-inhibit enzyme

• change enzyme from inactive to an active conformation

• stabilize active conformation

• induce translocation of an enzyme to the site of action

• no active site activators -> block active site

Bsp: PP1 de-inhibition = activation

heart muscle disease

PP1 dephosphorylates RyR -> no Ca2+ release

seal Ca2+ release equals healthy cell

Substrate specificity by holoenzyme (green)

Pathomechanism: CaMKII hyperactivity - SR Ca2+ leak -> increasing rate of heart muscle contraction, compensated hypertrophy, cardiac failure

Treatment: PDP - reduction of SR Ca2+ leak, dephosphorylation of RyR2 -> Partially restored Ca2+ homeostasis through PP1 de-inhibition

1. Approach: phosphopeptide library dephosphorylation followed by mass spectrometry (PLDMS):

selectivity determination on the peptide level

• synthesize random phosphopeptide library

2. Approach: phosphoproteomics in HeLa cell lysates:

selectivity determination on the protein level

Results:

• N-terminus + charged (basic AA)-> preference for PP1 but not PP2a (prefers acidic AA)

• Both phosphatases prefer phosphoThreonine gets dephosphorylated first

• PP1c contributes to selectivity, PP2A requires holoenzymes

• PP1 and PP2A show substrate specificity on the catalytic subunit and the holoenzyme level