TREMBLAY - LECTURE 2 Flashcards
characteristics of protein modifications
There are > 1000 protein modifications
mostly non reversible chemical modifications
small numbers are enzymatic modifications; few of them are reversible
what are the amino acids that can be phosphorylated and how
protein kinase structures and function
~550 protein kinases encoded in the human genome (1.5% of our genetic diversity)
Protein kinases regulate at some level almost all cellular processes
: cellular proliferation, cell cycle, replication, translation
Kinase domain = a catalytic entity with one basic function
:transfer the gamma phosphate moiety from ATP to a hydroxyl moiety of serine/threonine or tyrosine residues
Half life = weeks in aqueous solution (stable phosphorylation modification)
Phospho-moiety is hydrolyzed in the cell by protein phosphatase
protein phosphorylation ancestry
Kinase superfamily is derived from 1 ancestral gene
Phosphatase arose from the same ancestor (conserved motif)
structure of PPPs
PPP (phosphoprotein phosphatase)
Largest and most well-studied family (PP1, PP2A, PP2B)
PP1: associated proteins from 20 subunits bound to PP1 = specificity
PP2A: scaffolding protein binds catalytic unit = core enzyme
Core enzyme + regulatory subunit = trimer = holoenzyme
Holoenzyme can recognize a specific substrate
Regulatory subunit provides specificity
Known for their broad substrate specificity
Involved in a wide range of cellular processes
Deal with large amount of phosphoserine in the cell
Phosphoserine (substrate) function could be diverse
protein tyrosine characterization experiment
Procedure: take casein (protein) and phosphorylate it with purified src kinase → radioisotope used with nuclear reactor to label casein → perform chromatography on it → add radioactive phosphorylated substrates → follow its dephosphorylation
Linked proteins found
CD45: tyrosine phosphatase
marker for immune cells; to modulate T cell receptor
2nd domain: to protect active domain by recognizing oxidative effect
PTPase 1B: active enzyme
Kinase: many motifs at the surface and some conserved in kinase
PPM1
PPM1 (Metal-dependent Protein phosphatase)
Includes PP2C
Characterized by their requirement for metal ions for catalytic activity
Asp phosphatase
Asp phosphatase (Aspartate based phosphatase)
Includes FCP, SCP
Characterized by their catalytic mechanism involving an aspartate residue
characteristics of PTPs and signature motif
Function as a negative/positive regulator
Differentiation, proliferation, motility, metabolism, cell growth
Inhibit, but could also activate the effects of protein tyrosine kinases
Conserved catalytic domain of 240 amino acids
It maintained structure and the pocket for P-Tyr
Signature motif: [I/V]HCXAGXXR[S/T]
C: for nucleophilic attack
If there is this motif in the protein, 100% tyrosine phosphatase
emergence of PTP
Emerged before kinase
Cysteine can bind heavy metals which were abundant in the beginning of life due to volcanic eruptions
Phosphatase was used to bind heavy metals to protect life from them
PTP expression in human tumors
Tumor suppressor = dephosphorylate catalytic site, oncogenes = C-terminus
Both = dual specific phosphatase: dephosphorylate Ser/Thr/Tyr or sugar/lipid
types of PTPs
- Dual & atypical
MTMR: recognizes lipids
PRL (oncogenic): not very active as a phosphatase, pseudo catalytic domain that controls amount of Mg2+ coming into the cell
PTEN (tumor suppressor): most cancers have PTEN mutation - P-tyrosine specific
- Functions: remove phosphate moieties from
Phosphor proteins, lipids, sugar, other metabolites
Example of PTP inhibiting or activating signalling
- c-src = the proto-oncogenic form of viral v-src
- active = phosphorylated on Y416 in the catalytic domain
PTPs phosphate removal inhibit kinase activity
Use PTP-SHP-1 or PTPN13 (shut down src and activate kinase) - inactive = phosphorylated on Y527 in the C-terminal tail = negative regulator
PTPs phosphate removal activate kinase activity from an inhibitory site
PTPN2/TCPTP or PTP-SHP-2
the black plague and how it works
Yersinia pestis
Bacteria that causes black plague
Injectisome: makes a hole on the skin of the bug (tick) and it bites the next host and bacteria gets transferred
Inhibits immune cells and apoptosis
Yop H (transferred via injectisome) = phosphatase that dephosphorylate tyrosine on membrane and protein-protein interaction
structure of PTP1B
Structure
Dual specific: recognize P-Ser and P-Tyr (larger pocket for Ser)
PTP-loop: where cysteine is found
WPD-loop: pocket where PD loop close on it to stabilize dephosphorylation
mechanism of PTP1B
- Cys 215 and Gln 262 make covalent link and let go phosphate
- Phosphate stays on cysteine after taken away from protein
- Asp 181 and water from Gln 262 let go of phosphate from cysteine
induction of mutation and PTP1B
Can inactivate phosphatase, but still can recognize the substrates
Substrate trapping = radioactive phosphate on ATP to find out the substrate
key features
types of RPTs
RPTK1 (kinase): ligand binding induces dimerization and cross phosphorylation, which activates enzymatic activity = activate signalling
RPTP1 (phosphatase): transmembrane receptor dimerizes and activates phosphatase activity = inhibits signalling
PTP substrate identification using substrate trapping
Asp 181 on PD loop
Clamps substrate to enzyme; add alanine instead
Cys 215 on catalytic motif
Change it to Serine; can’t dephosphorylate
Mutation: no reaction but pocket exists
substrate: Jak2
PTP1B D181A: catalytically inactive but retain its ability to bind substrates
When changes from aspartate (D) to alanine (A): recognizes Jak2, but attaches to it
How to map the PTP1B binding site on cortactin
Tyr-phosphatase in the tube: not specific, but in cell: very specific and localized
Only dephosphorylated one doesn’t show up in the western blot
Where phosphatase resides!
PTP1B as target in metabolic diseases and cancer
Insulin receptor signal transduction
Insulin induces phosphorylation of receptor which activates IRS1 and causes biological effects downstream
PTP1B knockout mice resist diet induced obesity
