TREMBLAY - LECTURE 1 Flashcards
which amino acids are mainly affected by phospho and dephosphorylation?
serine/threonine and tyrosine
dephosphorylation can also lead to activation depending on the environment and the protein
what is the kinome
complete list of kinases in the genome
All kinases share a common catalytic core evolved from a common original kinase.
~120 protein tyrosine kinases (PTK)
~500 kinases
~30 Ser-Thr phosphatases catalytic subunits
>200 regulatory subunits
107 PTP (Protein tyrosine phosphatase)
how is functional diversity achieved by Ser/Thr phosphatases despite their lower number compared to kinases
achieved by forming large multiprotein complexes with regulatory subunits, which then recognize specific substrate
characteristics of tyrosine phosphatases
Tyrosine phosphatases may have evolved before kinases
Sensitive to heavy metals and can bind to it
Metals inhibit the tyrosine phosphatase by binding to its catalytic site.
Diversifying by adding different adapting segments that bind other phosphorylation and glycosylation sides.
what is the basic function of the kinase domain
Transfer the gamma phosphate moiety from ATP to a hydroxyl moiety of a serine/threonine or tyrosine residues
what are the two major types of phosphate linkages
Ester bonds: -O–P link between phosphate and hydroxyl group on Ser, Thr, Tyr.
Stable even in test tubes unless exposed to tons of phosphatase
Amide link: -N–P linkage seen in Phosphoproline/Phosphohistidine.
Unstable and Sensitive to PH. During SDS-PAGE, pH changes during gel electrophoresis break these bonds.
Eliminate the Amide bonds, hard to identify.
what are the domains that recognise and bind phosphorylated residues
SH2 (src homology 2) domain: binds phospho-tyrosine
Binds pYEEI ligands
PTB domain: binds phosphor-tyrosine
Less important. Shc and IRS-1 NPXpY ligands
14-3-3 domain: binds phosphoserine/ pSer ligands
FHA domain: recognize Phosphoserine
WD40 repeat domain, cdc4 b-trcp: di-phospho-peptide motif
Assembly of these domains allows for cascading signal transmission with high specificity.
what is a pseudo protein tyrosine phosphatase
Inactive enzymes that link p-tyrosine
Resemble active phosphatases but lack catalytic activity
kinase catalytic domain characteristics
All protein kinase shares a conserved catalytic domain and Catalytic mechanism
There are binding sites for Mg2+ Ions, which hold the ATP phosphate groups in the catalytic pockets.
ATP contains three phosphates
Only the γ-phosphate is transferred in kinase reactions.
features of phosphorylation signal cascades
Cascade events can be integrated
In response to different cellular stress, different kinases sense it in different ways but act on the same protein sites 🡪 e.g. Phosphorylate Ser51 residue of eIF2alpha and inhibit protein synthesis.
Distinct Regulatory domains but similar catalytic domains.
Cascade events can be branched.
E.g. AKT/PKB pathways
Have multiple downstream substrates.
Trigger different outcomes (e.g. Apoptosis, proliferation, angiogenesis).
Activate additional kinases, further branching the signal.
how was RSV discovered (Rous Sarcoma Virus)
Peyton Rous discovered the agent which was then identified as RSV (Rous Sarcoma Virus)
This virus genome encodes a constitutively active tyrosine kinase
Tony Hunter identified phosphotyrosine as a marker of transformation
He used Radioactively labeled ATP and cell extract of src-transformed NIH 3T3
In RSV-transformed cells, a new phosphoprotein spot appeared in the 2D electrophoresis gel.
Correlate with kinase activity 🡪 src is a kinase and recognize tyrosine
what is the structure of the Src kinase
The proto-oncogenic form of the viral v-SRC
phosphorylated on Y416 in the catalytic domain when active
phosphorylated on Y-527 in the C terminal tail when inactive.
Domain of src kinase:
Kinase (SH1) domain:
Contains catalytic site
Transfers γ-phosphate from ATP to substrate tyrosine residues
SH2:
Binding site for phosphorylated tyrosines.
Can bind to the phosphorylated C-terminal tail of src itself
Prevent the catalytic domain to be active.
SH3: Binds proline-rich motifs.
Myristate Palmitate (SH4:not widely used): Fatty acids binding.
conformations of inactive vs active Src
Inactivated Src
Y527 phosphorylated, bound by SH2 domain
SH3 binds the proline motif.
Kinase domain is inaccessible to ATP
Active SRC
Y527 dephosphorylated.
Open conformation allows substrate binding in the catalytic pocket
Other kinases can also phosphorylate Src’s y527
characteristics of receptor tyrosine kinases (RTKs)
RTKs are transmembrane proteins with intracellular kinase domains
Ligand binding leads to dimerization and then triggers transphosphorylation of kinase domains.
Ligands are typically growth factor molecules
Dimerization enables transphosphorylation of the kinase domain
After adding EGF, a new phosphotyrosine spot appears in the Electrophoresis gel.
RTK has various structures
Some RTKs are inactive until dimerized (e.g., most classic growth factor receptors).
Others, like the insulin receptor, are already dimerized in the absence of ligand.
Stay together by disulfide bond
characteristics of insulin receptors
Insulin receptor is a heterotetramer composed of two alpha-subunits and two beta-subunits
The receptors exist in a dimer conformation but remain inactive until insulin binding
Insulin binding causes a conformational change, bringing beta-subunit kinase domains together and leading to autophosphorylation of two tyrosine kinase domain
Autophosphorylation of the tyrosine 1158, 1162, and 1163 in the kinase domain of the beta chain
When these 3 tyrosines are autophosphorylated, they relieve the blockage of the active sites 🡪 Enhance tyrosine kinase activity of insulin receptor.
characteristics of IRS1 (insulin receptor substrate 1)
Important in diabetes 🡪 widely studied
IRS1 (Insulin Receptor Substrate 1)
Plays a key role in mediating the intracellular effects of insulin
Harbors several important domains through which it activates a variety of downstream kinases
Docking signal proteins
PTB domain: Binds pY960 of the insulin receptor.
PI3 kinase binds IRS-1 and converts PIP2 🡪 PIP3, leading to activation of PKB (Akt)
Akt phosphorylates and inactivated GSK-3(Glycogen synthase kinase), which normally inhibits glycogen synthase.
Disruptions in this pathway contribute to insulin resistance and type 2 diabetes
Oncogenic Activation of Receptor Kinases
Ligand-independent activation due to mutations can lead to uncontrolled cell proliferation.
E.g. v-ErbB mutation dimerize all the time
Dimerization leads to autophosphorylation
Autocrine signaling cells produce their ligand and receptors
Overproduction of ligand leads to hyperactivation of receptors and continuous downstream signaling (often found in cancer).
Cysteines in receptor extracellular domains form disulfide bridges, stabilizing the proper structure for ligand binding.
If one cysteine is missing (due to alternative splicing or mutation), the receptor may form aberrant disulfide bonds with another receptor.
This leads to ligand-independent dimerization and constitutive activation even in the absence of ligand.
E.g. ErbB2 becomes constitutively active due to such structural changes.
Philadelphia Chromosome
Philadelphia Chromosome = Translocation between chromosomes 9 (ABL) and 22 (BCR).
Creates BCR-ABL fusion protein:
BCR is the dimerization domain.
ABL is a cytoplasmic tyrosine kinase.
Junction of dimerization and kinase domain
The fusion leads to constitutive dimerization of ABL, causing constant kinase activity and driving CML (chronic myeloid leukemia).
Can produce inhibitors of ABL, but the patients develop resistance over time.
Protein Kinases Summary
Serve as an exquisite molecular switch through phosphorylation
A large number of enzymes in multiple biological systems
The most targeted enzyme family
Globally over $105 billion for the pharma industries.
