Phosphorylation is a form of post-translational modification which are the covalent and generally enzymatic medication of proteins during or after protein synthesis changing the properties and can be reversible

-        PTMs make the proteome more complex

Phosphorylation is the addition of a phosphate group

-        While there are lots of PTMs phosphorylation received and lot of action as kinases are often overexpressed or mutated in disease so lots of inhibitors have been developed

Protein phosphorylation is a reversable process where phosphate from ATP is sterified to amino acids by protein kinases

-        generally it activates the protein activing as a molecular switch

-        phosphorylates serine and threonine

Protein kinase phosphorylates and protein phosphates dephosphorylates

Kinases and phosphatases

The balanced activity between kinases and phosphateaases provide sufen and often short lived modifications of proteins resulting in effective signalling events

-        <500 kinases in the human genome

o   90 are tyrosine kinases

o   56 are transmembrane recepotrs

o   Some have dual specificity

-        They have highly conserved motifs

o   Activation loop

o   Catalytic domain

o   Atp binding domain

-        Many kinase structions have been solved which aids inhibitor design

-        Serine/throine kinases are activated by second messengers

Protein phosphatases have diverse structures

-        147 in the human human genome

-        100 prtoein tyrosine phosphateases (PTPs)

-        37 are phosphotyrosine specific including 21 receptor like PTPs

-        65 have dual specificity

Kinases vs phosphatases

Athough all the kinases have evolved from one ansestorial kinase gene the PP and PTP gene family do not share structural or sequence homologies

While phosphatases biochemical functions have been well studied the substrates of specific phosphates remain largely unknow

Phosphatease recognise and catalyse a wider arraty and substrates

While the phosphatases show greater variety the reaction is well conserved and does not require energy

Phosphatases targeting substrates

Module bases – targeting domains mediate cellular locallisation and substrate recruitment

SLiM-mediated – small linear motifs to dock into a substrate binding poclet

Regulatory subunit-assisted -phosphateases can bind non covalently to regulatory subunits or scaffold protein which mediate substrate docking

Role in insulin secretion

Insulin regulates the process of storing nutrients in forms that can be used as energy sources during periods of fasting

Insulin is an anabolic hormone that promotes proper metabolism, energy balance and maintenance of normal body weight

The binding of insulin

-        Activates intrinsic tyrosine kinase activity of receptor (IRK)

-        Which phosphorylates tyrosine residues of target proteins such as insulin receptor substrates

-        Which propagate three major signalling pathways

o   P13K

o   MAP kinase

o   Cb1/cAP pathway

P13K cascade is activated by the IRS protein which functions as a scaffolding protein to trigger signalling pathways

-        All proteins that bind to IRS contain SH2 domain

Control of insulin signalling

Can be activated by autoregulation, downstream components inhibit upstream elements

mTOR-Raptor complex inhibits P3 and phosphorylates IRS and GRB10 to inhibit the effect of insulin.

Alternativly signals from unrelated pathways can inhibit insulin signalling.

Insulin Resistance

Insulin resistance is the failure of ordinary, levels of insulin to activate the metabolic actions

-        Closely related to obesity and type 11 diabetes

Attributed to factors released from adipose called FFA, glycerol, hormones, and pro-inflammatory cytokines, which activates IRS kinases which phosphorylate IRS on serine which negatively effects insulin signalling

-        This makes type II very hard to treat as those with obesity have chronic low-grade inflammation and insulin resistance due to excess adipose tissue releasing the factors

IRS kinases can be divided into two groups

-        Mediators of insulin signalling which mediate proper insulin response by negatively regulating IRS protein upon prolonged insulin stimulation  

o   mTOR/S6K1

o   MAPK

o   PKC𝜁

-        Non-mediators of insulin signalling which are unrelated to insulin pathways such as inflammatory cytokines, fatty acids, amino acids, and ROS

o   GSK-3β

o   IKKβ

o   JNK

o   mPLK

o   AMPK

A network of serine kinases are involved in the down regulation of insulin signalling, in obesity and diabetes a network of serine kinases are activated that target IRS proteins

In grey are the kinases in the insulin signalling pathway

In purple there are the kinases activated by inflammatory cytokines, FFAs, amino acids, ROS

The list of IRS kinases and potential serine phosphorylation sites implicated in the development of insulin resistance is growing (NOTE – work by phosphorylating serine which inactivates the process)

-        PH domain is critical for IR-IRS interactions

-        PTB domain which interacts with the juxtamembrane domain of IR

-        Phosphorylation events lower down interact with SH2 domains of effector proteins stopping them from acting as scaffolding proteins

Phosphorylation events affect IRS in two ways, preventing IRS from binding to the receptor so it cannot get activated or stop it from binding to downstream effector proteins

In pathophysiological conditions, inducers of insulin resistance activate several serine kinases such as IKKβ, KJN, ERK, S6K, that phosphorylate IRS 1 and IRS 2 on inhibitory sites in an uncontrolled manner

Regulation of retinoblastoma (Rb) protein activity

When RB is hypo-phosphorylated it inhibits E2F transcription factors which prevents the expression of genes involved in G1 to S progression

-        Very important in regulation of cell division, mutations can cause tumours and cancer

The level of phosphorylation is closely related to the cycle

-        When Rb is in hypo-phosphorylation it is the M or G1 cycle

-        When RB is hyper-phosphorylated it stops inhibiting E2F transcription factors so the cell cycle can continue

Antigrowth factors such as TGFβ blocks the cell cycle by inhibiting Rb phosphorylation keeping it in a hypo state so the cell cycle cannot move from G1 to S

-        TGFβ blocks the cyclin CDK complex responsible for Rb phosphorylation