Protein Kinases

Amino acid sequence motifs often identify or suggest functional characteristics whereas amino acid domains typically relate to structural units with specific functions. Domains are usually encoded by a single exon.

Phosphatases and protein kinases are typically found as part of a signalling cascade which amplify signals. Kinases phosphorylate proteins whereas phosphatases reverse kinase actions. The human genome encodes around 520 kinases and 150 phosphatases. Serine/threonine kinases and tyrosine kinases are most well studied. Histidine kinases are much rarer. Both structural and mechanistic conservation is shown in this family.

Bioinformatics has identified over 500 genes encoding kinases and has helped to identify key catalytic residues. Sequence alignments can be used to reveal evolutionary and regulatory similarities. Primary sequence alignment is shown between the catalytic domains of PKA (res 33 to 283) and PknB (residues 1 to 266). On an evolutionary tree, kinases are found clustered in specific branches. PKA, PKC and PKG for example are found in the same branch due to all being regulated by allosteric binding eg by calcium.

Protein kinase A aka cAMP dependent protein kinase:

PKA structure: Heterodimer of 2 regulatory (R) subunits and 2 catalytic (C) subunits. As cAMP increases it binds the R subunit inducing a conformational change in which the complex dissociates releasing C subunits (active form). Binding site characterised by GxGxxG sequence motif

Glucagon is released in response to low blood glucose levels. It activates protein kinase A. PKA phosphorylates glycogen synthase a, converting it to its inactive form glycogen synthase b, stopping glycogen synthesis. PKA also phosphorylates inactive glycogen phosphorylase b converting it to its active a form, leading to breakdown of glycogen to release glucose. 1 glucagon molecule causes the breakdown of 10⁸ glycogen molecules.

General PKA mechanism:

• ligand binds GPCR

• Activates adenylyl cyclase (ATP → cAMP)

• cAMP activates PKA

• cellular response

Kinases share structural features. Their basic catalytic mechanism is conserved and they have similar structured active sites with a characteristic arrangement of key residues eg DFG motif at active site. Lysine or arginine stabilises the transition state.

General mechanism:

• ATP binds kinase active site

• substrate binds active site

• γ-phosphate transferred from ATP to Ser/Thr/Tyr

• sub released from kinase

• ADP released from active site

Activation of kinases:

Often occurs by unmasking active site. Mechanisms of unmasking include ‘activation loop’ displacement, or via ‘pseudo-substrate’ domains

Activation loops are found in many kinases and they physically block access to the active site. An event such as phosphorylation of the activation loop occurs resulting in a conformational change → active site unblocked. This is sometimes referred to as a ‘priming’ event.

Protein Kinase C:

The PKC superfamily contains homologous proteins made up of domains. Conformational changes to PKS are brought about by binding of calcium ions and diacylglycerol (DAG) which activates the kinase domain → signal transmission.

A key feature of PKC is its pseudosubstrate domain, a short amino acid sequence lacking serine or phospho-acceptor residues. This region is able to bind the substrate-binding cavity to keep the enzyme inactive. DAG interacting with the membrane by binding the C1 domain causes the domain to be released from the catalytic site, activating the enzyme. Both calcium ions and DAG must be present.

The pseudosubstrate domain is found in very close proximity to the domain being phosphorylated. When inactive the active site is bent around the pseudosubstrate domain meaning it can’t be accessed.

Target specificity:

The sequences around the phospho-acceptor site of kinase targets are similar. This means bioinformatics can be used to predict the structure of these sites in other proteins. For example many kinase targets include arginine xx serine motif.

Kinases phosphorylate their targets to regulate target enzymatic activity. This may change the protein structure and also as a result could change the protein’s conformation and ability or selectivity in interactions.

Phosphorylation by a kinase can change a protein’s:

• localisation

• interactions eg increasing or decreasing affinity, or mediate selective interactions

• half life

• sensitivity to signals