Biol729 – deciphering the kinome

Phosphorylation screening

A number of techniques exist for identification of substrates for protein kinases by in vitro phosphorylation (note. there are ~ 20,000 phoshoproteins in a cell with serine, threonine or tyrosine phosphorylated in the ratio of ~ 1,800:200:1).

These screens usually require purified, active protein kinase and this may not always be easy to produce.

Expression in bacterial or other cells may not be possible and upstream activating protein kinases may not be known.

Purifying the protein kinase from mammalian cells may be productive, but contaminating protein kinases may be a problem.

A major problem with phosphorylation screening is the ability of kinases to phosphorylate ‘inappropriate’ substrates in the absence of the normal in vivo regulatory constraints, producing false-positive substrate identifications.

In vitro phosphoryation

Classical in vitro lysate phosphorylation has been coupled with mass spectrometry for kinase substrate tracking and elucidation (KESTREL).

Cell lysates are incubated with purified active protein kinase and the resulting phosphoproteins are separated by gel electrophoresis and then identified by tryptic mass fingerprinting.

MALDI-qTOF MS analysis of a tryptic digest. Letters a and b indicate peaks of low signal intensity corresponding to the phosphopeptides LDLIDEEEDpSEEDGDSMR and VLpTPLQVQGEGEGR, respectively.

Historically, in vitro protein kinase assays used radioactive ATP. Now, fluorescently labelled peptides can be used. The resulting phosphorylated peptides bind to an IMAP reagent (a trivalent metal ion with a high affinity for phosphates), causing an increase in the polarization of the fluorescence. For kinases of unknown specificity, arrays of different peptides in microwell plates are available.

Phage-based assays

Phage expression libraries can be used to produce a library of potential kinase substrate proteins that can be incubated with a purified kinase.

The library is plated out on a bacterial lawn and the phage plaques are transferred to a nitrocellulose membrane which is blocked and then incubated with purified kinase and [- 32 P]ATP.

Autoradiography indicates which plaques are phosphorylated. The cDNAs from replicated plaques are sequenced and the proteins they encode are verified as substrates.

Phosphorylation screening for cloning of protein kinase substrates.

Peptide and protein array screens

The yeast kinome has been assayed using protein chips. The chips were probed with 119 glutathione-S-transferase (GST)-tagged proteins representing nearly all the 122 yeast protein kinases.

Protein chip fabrication and kinase assays.a, PDMS (a disposable silicone elastomer), is poured over an acrylic mould. After curing, the chip containing the wells is peeled away and mounted on a glass slide. The next step includes modification of the surface (incorporation of a cross-linker) and then attachment of proteins to the wells. Wells are blocked with 1% BSA before protein kinase, [32P] -ATP and buffer are added. After incubation for 30 min at 30 °C, the chips are washed extensively and exposed to both X-ray film and a phosphoimager. b, An enlarged picture of the protein chip.

Phosphoimager signals are transformed into fold increases. a, Signals of 119 kinases in 4 reactions are shown. The fold increases ranges from 1 to 1000-fold. The numbers on the axis indicate the kinase that was analysed. b, To determine substrate specificity, a ‘specificity index’ was calculated. Examples of kinase specificity are shown where SI > 3.

The 3-Dimensional flow-through PamChip® array technology A) Electron microscope image showing the porous structure of the anopore sheets, the solid support in which the peptides are immobilized. B) Each peptide position is called a spot. C) Up to 400 peptide spots are present per microarray. D) A chip comprises 4 or 96 peptide arrays. E) Flow-through fluidics using 25 L volume sample. F) Kinetics: during the incubation multiple fluorescent microscopic images are taken.

Peptide arrays allow rapid characterization of the preferred primary sequences for a protein kinase. A typical array may consist of peptides based on known phosphorylation sites or of peptides of random sequence.

Protein kinase assay. A peptide array comprising 144 different phosphosite peptides is incubated with purified kinases (Csk or Yes shown) or a cell lysate comprising multiple kinase activities (not shown). Time curves of signals plotted versus time are generated for each of the 144 peptides.

General problems with these techniques are non-specific absorption of the liquid-phase proteins to the chip or incorrect folding rendering proteins non-functional.

Functional knockouts to investigate kinases

This class of technique involves obliteration of protein kinase activity and subsequent investigation of the consequences.

Mice with genetic deletions or knock-in mutations of protein kinases and cell lines derived from them are valuable tools.

RNAi technology offers a convenient means of depleting specific proteins. Potential drawbacks include incomplete depletion and non-specific effects. Pools of pre-characterised siRNAs for each kinase are now available on a kinome-wide scale.

Increasing numbers of specific small-molecule inhibitors of protein kinases are available and can provide rapid preliminary tests for the involvement of a kinase of interest in a particular process.

1. Mouse development begins when the egg is fertilised by a sperm. 2. The mass of dividing cells differentiate and forms a blastocyst, a hollow sphere of cells containing an inner cell mass of embryonic stem (ES) cells.3. ES cells have the ability to replicate and divide in vitro. Thus ES cell lines can be established by removing cells from the blastocyst.4. To generate transgenic mice, gene sequences are introduced into the genome of the ES cells. Those cells containing the new/mutated gene are selected and grown. 5. Transformed ES cells are transferred back into a blastocyst which is implanted into a surrogate mouse.6. Progeny from the surrogate mouse that are heterozygous for the new gene are mated to generate homozygous mice.

Generation of transgenic mice

Generation of ERK5 knockout mice. A) Use of a targetting vector to delete exons 4 and 5 of the murine ERK5 gene by addition of a neomycin selection cassette. B) ES cell DNA digested with Hind III and Mfe I, and Southern blotted using a probe 3' to the targetting vector. The wt 9.5 kb fragment and targetted 3.3 kb fragment are indicated. C) DNA from E9.5 embryos digested with Hind III and Mfe I. Southern blotted as described in (B). D) Soluble protein from E9.5 embryos immunoblotted using antibodies to ERK5, MKK5, ERK1/2 or p38.

Short siRNA molecules unwind into single strand RNAs, which then combine with proteins to form a RISC [RNA-Induced Silencing Complex].

The RISC then captures a native mRNA molecule that complements the siRNA sequence.

If the pairing [native mRNA and siRNA piece] is essentially perfect, the native mRNA is cut into RNA fragments that aren't translated.

If the pairing is less than perfect then the RISC complex binds to the mRNA and blocks ribosome movement along the native mRNA also halting translation.

(A) Protein kinase Cdomain structure and sequences in the protein kinase C mRNA against which siRNA species were prepared. (B) adipocytes were trans-fected with the four siRNA species shown in (A) or with siRNA directed against both Akt1/PKB and Akt2/PKB. 3H-deoxy-glucose transport was then assayed. Only the Akt-directed siRNA inhibited insulin action with statistical significance (*P<0.05).

Depletion of protein kinase C by siRNA fails to attenuate insulin action on deoxyglucose

transport.

Increasing numbers of specific small-molecule inhibitors of protein kinases are available and can provide rapid preliminary tests for the involvement of a kinase of interest in a particular process.

Summary

http://www.phosphosite.org/

http://scansite.mit.edu/

References

Zhu, H et al., (2000) Nature Genetics 26, 283 - 289 (2000) - Analysis of yeast protein kinases using protein chips.

Johnson, S. A. & Hunter, T. (2005) Nature Methods 2, 17-25. Kinomic methods.