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): 1912ô€‚±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 ô€‚± 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 ô€‚±
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
Always on Time
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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 ô€‚± 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 ô€‚³ Gain of functionô€‚´
(New function ) or ô€‚³ Loss of Functionô€‚´ ( 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 ô€‚± 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, 168ô€‚±174. This gives an example of the applied use of
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 ô€‚± protein interactions network .
Phage display fusion libraries is a method to study the protein ô€‚± 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 .
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): 7382ô€‚±7.
4 Rigaut G., et al. (1999). "A generic protein purification method for protein complex characterization and proteome exploration".
Nature Biotechnology 17 (10): 1030ô€‚±1032.#
5 Smith GP (1985). "Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface".
Science 228 (4705): 1315ô€‚±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
6 Somanath PR, Razorenova OV, Chen J, Byzova TV (March 2006). "Akt1 in endothelial cell and angiogenesis". Cell Cycle 5
In recent years , our knowledge of phosphorylation ô€‚± 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
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 .