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Phosphorylation is of import in cell signaling in populating being, and it requires the action of protein kinase. In life cell, there are a batch of regulative procedures and tracts commanding 1000 of functional protein and metabolic reaction. Therefore, it is indispensable for us to understand the relationship between different kinases and substrates in order to decode the whole signal transduction system inside our cell.

However, it is non a easy undertaking for scientists to catalog the protein phosphorylation due to the immense figure of kinases and phosphoproteins may be expressed in cell. Specifying the kinase complement of the human genome is the Kinome, which consists of 518 kinases1. To placing physiological substrates of protein kinase, individual technique is non sufficient to give a accurate and comparable consequence, therefore, modern-day techniques would affect combination of different techniques to analyze the phosphorylation in life cell.

Background and aim:

When we try to understand the protein phosphorylation, we may inquire two inquiries of what and which. What substrates and sites does a peculiar kinase phosphorylate? And which kinase is responsible for phosphorylating an identified phosphorylation site? However, replying these inquiries is non an easy undertaking. Normally, scientist would look into protein phosphorylation in vivo utilizing different research lab techniques. Although it is really of import in the beginning of the survey of a certain kinase, the information is non detail plenty and it still understanding of the whole phosphorylation web. As a consequence, a combination of different techniques is required in order to undertake the job. This paper reviewed a group of modern-day combination of techniques which are used in protein phosphorylation.

Major techniques used:

In this reviewed paper, the author divided the techniques into three chief 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 wish to place

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

genome ” . Science 298 ( 5600 ) : 1912oˆ‚±34.

the substrate of it. Substrate to kinase techniques turn to the inquiry of which

kinase phosphorylates my substrate. Finding out the relationship between two

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

kinase to or from substrate techniques. Three of them are all of import in the survey of

protein phosphorylation.

Main Content:

1 ) Kinase to substrate

A ) Phosphorylation testing

There are two chief types of phosphorylation showing which are library or

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

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

possible and targeted substrate. While the Candidate substrate showing is that we

hold several possible substrates for the kinase interested and we are traveling to corroborate

the possible substrate. In phosphorylation showing, it requires purified and active

kinase obtained from stirred mammalian cell, but non bacterial cell as the

physiological status in bacterial cell is different to that of homo. However, there

are 1000 types of kinase in cell lysate and it is hard to aim or concentrate to the

phosphorylation that we are interested oˆ‚± taint. Besides, false- positive

substrate may besides be found as some substrates may be phosphorylated in vitro but non

in vivo ordinance.

Example 1: In vitro protein phosphorylation

Cell infusions are incubated with high degree purified, active kinase of involvement,

besides with radioactive Mg2+- [ -32P ] ATP, leting the phosphorylation to happen. The

phosphoproteins are so separated by gel cataphoresis and identified by mass

spectroscopy. There are three possible jobs. First, the cell extracts contain all

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

upon incubate with ATP, bring forthing background phosphorylation. Second, many

kinase phosphorylate protein in vitro, but non in physiological substrate ( False oˆ‚±

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

devouring. However, we can cut down the incubation clip with Mg2+- [ -32P ] ATP,

with higher concentration of kinase interested can cut down the Numberss of unwant

phosphorylation. Using Mn2+- [ -32P ] ATP alternatively of Mg2+- [ -32P ] ATP can besides

cut down unwant phosphorylation as non all kinase are able to utilize Mn2+ ATP expeditiously

in cell.

Example 2: Bacteriophage oˆ‚± based checks

Bacteriophage plaques are transferred to a lawn or membrane and so incubate with a

purified kinase and [ -32P ] ATP. We can utilize the autoradiography to find the

strongly phosphorylate phage. The complementary DNA is isolated from the phage and the protein

encoded is sequenced. In this check, phosphospecific antibody can be used to observe

phosphorylation as it can adhere to or observe phosphorylation induced alteration of protein.

Protein chips2 is used in order to find the presence or sum of protein and

phosphorimager is besides used as it make usage of radiation of different wavelength to

record a two-dimensions image, leting a quantitive comparing of the extent

phosphorylation of a peculiar substrate by kinase.

B ) Genetic Screening

In the Genetic showing, scientist would set up a weak oˆ‚? Addition of functionoˆ‚?

( New map ) or oˆ‚? Loss of Functionoˆ‚? ( less/no map ) mutation phenotype for

kinase of involvement. Then we screen 2nd site mutant that suppress the phenotype

and we target that 2nd mutant site and the merchandise of that cistron in the screen can

be tested as substrate of kinase interested.

C ) Functional smashers to look into kinase

Scientists knockout the cistron or stamp down the activity of the kinase of involvement by

RNA intervention or specific inhibitor to do the cistron no longer to work

usually and look into the effects of the suppression and functional smashers.

Then, we can detect the map and substrate of the kinase through the

annihilation of the kinase activity. Although this general attack merely identifies

engagement of a kinase in a peculiar tract, it is really of import in the beginning

of the survey of a certain kinase. Other techniques are still required to show

direct phosphorylation of a peculiar substrate.

D ) ATP parallel oˆ‚± sensitive kinases

In this attack, the ATP – adhering pocket of kinase interested is mutated to a

glycine. This mutant signifier of interested kinase that is able to utilize an ATP-analog is

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

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

web for the ErbB receptors utilizing protein microarrays ” . Nature 439, 168oˆ‚±174. This gives an illustration of the applied usage of

protein microarrays.

infusion. Merely the phosphoproteins that phosphorylated by interested mutant kinase

are detected as merely the mutant signifier kinase can use the radioactive ATP-analog to

make phosphorylation and generate phosphoproteins even in the presence of other

kinase. As a consequence, it can demo the kinase substrate relationship straight and it can

aid to extinguish all the background phosphorylation in the cell infusion. However, if

the clip of incubation clip of ATP parallel addition, the radioactive phosphate in

parallel may leak to the pool of cellular ATP.

2 ) Kinase to or from substrate

A ) Physical Association

Tandem affinity purification, yeast two – intercrossed screen3 and phage show

merger libraries all are the illustrations of physical association. Tandem affinity

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

extremely purified composites by consecutive purification by consecutive purification against

different protein tickets ( e.g. TAP ticket ) . As the sensitiveness of the mass spectroscopy is

really high, it can be used to place the proteins associated with a peculiar kinase or

substrate. This attack can be used to look into a peculiar kinase of involvement, and

this has been used to decode the planetary protein oˆ‚± protein interactions web.

Bacteriophage show merger libraries is a method to analyze the protein oˆ‚± protein

interaction and it can assist scientist to happen out the direct interaction of proteins. Gene

coded for possible proteins is introduced to bacteriophages, leting the introduced

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

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

shows a protein that binds to one of those marks on its surface will stay while

others are removed by rinsing. Those that remain can be eluted and so identified.

B ) Bioinformatics

Bioinformatics designation of the kinase responsible for phosphorylation a

peculiar site are non wholly dependable but offer a speedy anticipations that possibly

3 Joung J, Ramm E, Pabo C ( 2000 ) . “ A bacterial two-hybrid choice system for analyzing protein-DNA and protein-protein

interactions ” . Proc. Natl. Acad. Sci. U.S.A. 97 ( 13 ) : 7382oˆ‚±7.

4 Rigaut G. , et Al. ( 1999 ) . “ A generic protein purification method for protein complex word picture and proteome geographic expedition ” .

Nature Biotechnology 17 ( 10 ) : 1030oˆ‚±1032. #

5 Smith GP ( 1985 ) . “ Filamentous merger phage: fresh look vectors that display cloned antigens on the virion surface ” .

Science 228 ( 4705 ) : 1315oˆ‚±1317.

tested by other techniques.It is a powerful technique in replying the inquiry of

what sequence of polypeptide is more phosphorylated? The nucleus attack of

bioinformatics is specifying sequence penchant of kinase. A library of peptides is

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

fixed figure residue place, organizing a bunch of unknown amino acid sequence

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

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

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

most likely is the substrate.

3 ) Substrate to kinase

A ) Kinase renaturation testing

Candidate kinases or cell infusions are resolved on an SDS gel incorporating a

substrate of involvement within it. As protein unlike the nucleic acid, they have changing

charges and form and they are non migrate at similar rate. They have to be denatured

and coated with a negative charge for gel cataphoresis in order to divide different

kinase in cell harmonizing to their size. After gel cataphoresis, the kinases are

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

so detected by the autoradiography and identified by mass spectroscopy. However,

this techniques can non be used to look into all kinase as non all kinase can be

renature to make phosphorylation.

AKT / PKB is a serine / threonine protein kinase that plays a cardinal function in multiple

cellular procedures such as glucose metamorphosis, cell proliferation, programmed cell death,

written text and cell migration6. In the paper, it shows a timeline of find of

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

agencies by which they have been identified has changed over the old ages with engineering

progresss. In the early phase, individual chromatographic separation and in vitro

phosphorylation technique is needed to place 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 designation in the ulterior phase with engineering progresss. As a consequence, 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

( 5 ) : 512oˆ‚±8.


In recent old ages, our cognition of phosphorylation oˆ‚± based signaling webs

has advanced tremendously, come oning at a fast gait. Many mathematical theoretical accounts

can be computed, bring forthing a batch of accurate consequences and informations. However, jobs

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

remained and more integrating of research lab techniques is still needed to turn to the



By and large, this paper is good as it is really enlightening, supplying a batch of

modern-day combination of techniques to look into the protein phosphorylation in

cell. Besides the author can group different techniques in a good mode and split them

into different classs consistently. However, there are still room for

betterment. There is no diagram or chart to exemplify the rule of different

techniques, so this paper is a small spot dullard for readers. Besides, this paper is really

hard to understand, utilizing a batch of hard words and including many techniques merely

with the name, without any farther account. Therefore, it is non a easy undertaking for

readers to understand all the content in this paper.

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