Kinomics Methods For Deciphering The Kinome Biology Essay

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Phosphorylation is important in cell signaling in living organism , and it requires the action of protein kinase . In living cell , there are a lot of regulatory processes and pathways controlling thousand of functional protein and metabolic reaction . Thus, it is essential for us to understand the relationship between different kinases and substrates in order to decipher the whole signal transduction system inside our cell .

However , it is not a easy task for scientists to catalog the protein phosphorylation due to the huge number of kinases and phosphoproteins may be expressed in cell. Defining the kinase complement of the human genome is the Kinome , which consists of 518 kinases1 . To identifying physiological substrates of protein kinase , single technique is not sufficient to give a accurate and comparable result , thus , contemporary techniques would involve combination of different techniques to study the phosphorylation in living cell .

Background and objective :

When we try to understand the protein phosphorylation , we may ask two questions of what and which . What substrates and sites does a particular kinase phosphorylate ? And which kinase is responsible for phosphorylating an identified phosphorylation site ? However , answering these questions is not an easy task. Normally , scientist would investigate protein phosphorylation in vivo using different laboratory techniques . Although it is very important in the beginning of the study of a certain kinase , the information is not detail enough and it still understanding of the whole phosphorylation network . As a result , a combination of different techniques is required in order to tackle the problem . This paper reviewed a group of contemporary combination of techniques which are used in protein phosphorylation .

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Major techniques used :

In this reviewed paper , the writer divided the techniques into three main groups :

Kinase to substrate ; Substrate to kinase ; Kinase to or from substrate . Kinase to substrate techniques mean that we have a known kinase and we would like to identify

1 Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (December 2002). "The protein kinase complement of the human

genome". Science 298 (5600): 1912􀂱34.

the substrate of it . Substrate to kinase techniques address the question of which

kinase phosphorylates my substrate . Finding out the relationship between two

proteins from a library of kinase and substrate without a specific target are called

kinase to or from substrate techniques . Three of them are all important in the study of

protein phosphorylation .

Main Content :

1) Kinase to substrate

A) Phosphorylation screening

There are two main types of phosphorylation screening which are library or

lysate screening and Candidate substrate screening . Library or lysate screening is that

we search for a substrate of a kinase interested from a library of substrate without any

potential and targeted substrate . While the Candidate substrate screening is that we

have several potential substrates for the kinase interested and we are going to confirm

the potential substrate . In phosphorylation screening , it requires purified and active

kinase obtained from stimulated mammalian cell , but not bacterial cell as the

physiological condition in bacterial cell is different to that of human . However , there

are thousand types of kinase in cell lysate and it is difficult to target or focus to the

phosphorylation that we are interested 􀂱 contamination . Also , false- positive

substrate may also be found as some substrates may be phosphorylated in vitro but not

in vivo regulation .

Example 1 : In vitro protein phosphorylation

Cell extracts are incubated with high level purified , active kinase of interest ,

also with radioactive Mg2+-[ -32P]ATP , allowing the phosphorylation to occur . The

phosphoproteins are then separated by gel electrophoresis and identified by mass

spectrometry . There are three potential problems . First , the cell extracts contain all

protein kinase expressed in cell and it would produce all hundred of phosphoprotein

upon incubate with ATP , producing background phosphorylation . Second , many

kinase phosphorylate protein in vitro , but not in physiological substrate ( False 􀂱

positive substrate ) . Third , purification of kinase and phosphoprotein is time

consuming . However , we can reduce the incubation time with Mg2+-[ -32P]ATP ,

with higher concentration of kinase interested can reduce the numbers of unwant

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phosphorylation . Using Mn2+-[ -32P]ATP instead of Mg2+-[ -32P]ATP can also

reduce unwant phosphorylation as not all kinase are able to use Mn2+ ATP efficiently

in cell .

Example 2 : Phage 􀂱 based assays

Phage plaques are transferred to a lawn or membrane and then incubate with a

purified kinase and [ -32P]ATP . We can use the autoradiography to determine the

strongly phosphorylate phage . The cDNA is isolated from the phage and the protein

encoded is sequenced . In this assay , phosphospecific antibody can be used to detect

phosphorylation as it can bind to or detect phosphorylation induced change of protein .

Protein chips2 is used in order to determine the presence or amount of protein and

phosphorimager is also used as it make use of radiation of different wavelength to

record a two-dimensions image , allowing a quantitive comparison of the extent

phosphorylation of a particular substrate by kinase .

B) Genetic Screening

In the Genetic screening , scientist would establish a weak 􀂳 Gain of function􀂴

(New function ) or 􀂳 Loss of Function􀂴 ( less/no function ) mutant phenotype for

kinase of interest . Then we screen second site mutation that suppress the phenotype

and we target that second mutation site and the product of that gene in the screen can

be tested as substrate of kinase interested .

C) Functional knockouts to investigate kinase

Scientists knockout the gene or suppress the activity of the kinase of interest by

RNA interference or specific inhibitor to make the gene no longer to function

normally and investigate the consequences of the inhibition and functional knockouts .

Then , we can discover the function and substrate of the kinase through the

obliteration of the kinase activity . Although this general approach only identifies

involvement of a kinase in a particular pathway , it is very important in the beginning

of the study of a certain kinase . Other techniques are still required to demonstrate

direct phosphorylation of a particular substrate .

D) ATP analog 􀂱 sensitive kinases

In this approach , the ATP - binding pocket of kinase interested is mutated to a

glycine . This mutant form of interested kinase that is able to use an ATP-analog is

used to phosphorylate substrate . This [ -32P]ATP analog is then added to the cell

2 Richard B. Jones, Andrew Gordus, Jordan A. Krall, and Gavin MacBeath (12 January 2006) "A quantitative protein interaction

network for the ErbB receptors using protein microarrays". Nature 439, 168􀂱174. This gives an example of the applied use of

protein microarrays.

extract . Only the phosphoproteins that phosphorylated by interested mutant kinase

are detected as only the mutant form kinase can utilize the radioactive ATP-analog to

do phosphorylation and generate phosphoproteins even in the presence of other

kinase . As a result , it can show the kinase substrate relationship directly and it can

help to eliminate all the background phosphorylation in the cell extract . However , if

the time of incubation time of ATP analog increase , the radioactive phosphate in

analog may leak to the pool of cellular ATP .

2) Kinase to or from substrate

A) Physical Association

Tandem affinity purification , yeast two - hybrid screen3 and phage display

fusion libraries all are the examples of physical association . Tandem affinity

purification4 works on the basic of affinity chromatography technique and it produces

highly purified complexes by sequential purification by sequential purification against

different protein tags ( e.g. TAP tag ) . As the sensitivity of the mass spectrometry is

very high , it can be used to identify the proteins associated with a particular kinase or

substrate . This approach can be used to investigate a particular kinase of interest , and

this has been used to decipher the global protein 􀂱 protein interactions network .

Phage display fusion libraries is a method to study the protein 􀂱 protein

interaction and it can help scientist to find out the direct interaction of proteins . Gene

coded for potential proteins is introduced to bacteriophages , allowing the introduced

gene to be expressed and displayed on the surface of phages5 . A purified kinase is

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immobilized and incubated with a phages library of potential substrates . A phage that

displays a protein that binds to one of those targets on its surface will remain while

others are removed by washing . Those that remain can be eluted and then identified .

B) Bioinformatics

Bioinformatics identification of the kinase responsible for phosphorylation a

particular site are not completely reliable but offer a quick predictions that maybe

3 Joung J, Ramm E, Pabo C (2000). "A bacterial two-hybrid selection system for studying protein-DNA and protein-protein

interactions". Proc. Natl. Acad. Sci. U.S.A. 97 (13): 7382􀂱7.

4 Rigaut G., et al. (1999). "A generic protein purification method for protein complex characterization and proteome exploration".

Nature Biotechnology 17 (10): 1030􀂱1032.#

5 Smith GP (1985). "Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface".

Science 228 (4705): 1315􀂱1317.

tested by other techniques .It is a powerful technique in answering the question of

what sequence of polypeptide is more phosphorylated ? The core approach of

bioinformatics is defining sequence preference of kinase . A library of peptides is

synthesized consisting of an invariant serine , theonine or tyrosine residue linked by a

fixed number residue position , forming a cluster of unknown amino acid sequence

population at random . A population of protein is phosphorylated by interested kinase

and they are isolated and sequenced , finding out the sequence that is preferenced and

the amino acid which are strongly favored . The strongly favored amino acid sequence

most probably is the substrate .

3) Substrate to kinase

A) Kinase renaturation screening

Candidate kinases or cell extracts are resolved on an SDS gel containing a

substrate of interest within it . As protein unlike the nucleic acid , they have varying

charges and shape and they are not migrate at similar rate . They have to be denatured

and coated with a negative charge for gel electrophoresis in order to separate different

kinase in cell according to their size . After gel electrophoresis , the kinases are

renatured , incubating [ -32P]ATP and start phosphorylation . Phosphoproteins are

then detected by the autoradiography and identified by mass spectrometry . However ,

this techniques cannot be used to investigate all kinase as not all kinase can be

renature to do phosphorylation .

AKT / PKB is a serine / threonine protein kinase that plays a key role in multiple

cellular processes such as glucose metabolism , cell proliferation , apoptosis ,

transcription and cell migration6 . In the paper , it shows a timeline of discovery of

select AKT substrates . Numerous substrates have been identified for AKT and the

means by which they have been identified has changed over the years with technology

advances . In the early stage , single chromatographic separation and in vitro

phosphorylation technique is needed to identify the substrate of AKT kinase . From

the timeline , we can see that more combination of techniques such as

phosphospecific substrate antibody and bioinformatics techniques have been used for

substrate identification in the later stage with technology advances . As a result , more

and more substrates of AKT have been identified with more and more combination of

techniques .

6 Somanath PR, Razorenova OV, Chen J, Byzova TV (March 2006). "Akt1 in endothelial cell and angiogenesis". Cell Cycle 5

(5): 512􀂱8.

Conclusion :

In recent years , our knowledge of phosphorylation 􀂱 based signaling networks

has advanced enormously , progressing at a fast pace . Many mathematical models

can be computed , producing a lot of accurate results and data . However , problems

of kinase or substrate with low specificity , require for adaptor protein are still

remained and more integration of laboratory techniques is still needed to address the

problem .

Critiques :

Generally , this paper is good as it is very informative , providing a lot of

contemporary combination of techniques to investigate the protein phosphorylation in

cell . Also the writer can group different techniques in a good manner and divide them

into different categories systematically . However , there are still room for

improvement . There is no diagram or chart to illustrate the principle of different

techniques , so this paper is a little bit bore for readers . Also , this paper is actually

hard to understand , using a lot of difficult words and including many techniques just

with the name , without any further explanation . Thus , it is not a easy task for

readers to understand all the content in this paper .