Human p21(CDKN1A)-activated kinase 4

Target ID:

PAK4A

Deposition Date:

Tuesday 22nd May 2007

Families:

Non-protein Kinase

Related Diseases:

CancerSignallingNeurobiology

Structure type:

protein-ligand

Download Coordinates:

2Q0N

Authors:

Filippakopoulos, P., Eswaran, J., Turnbull, A., Papagrigoriou, E., Pike, A.W., Von Delft, F., Sundstrom, M., Edwards, A., Arrowsmith, C.H., Weigelt, J., Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

2Q0N

tabs

Structure Details

PAK-4 belongs to the STE20 subfamily of Ser/Thr protein kinases and was the first member identified of the Group II family of PAK kinases (PAK-4, PAK-5 and PAK-6). Structurally, p21-activating kinases are characterized by an N-terminal p21 binding domain (PBD). Although PBD's of the Group II PAKs are quite different from those of the Group I PAKs, they still bind to activated Rho family members. However, Cdc42 mutants like Y40C which does not bind PAK-1, 2, and 3, retains PAK-4 binding activity suggesting that the interaction of these two proteins is considerably different. In contrast to group I PAK's, binding to activated GTPases does not stimulate PAK-4 kinase activity. This difference might be explained by the lack of an identifiable inhibitory switch domain (ISD) in PAK-4 and other members of the group II family. The ISD has been shown to lock PAK-1 in an inactive state. This inhibition is released by binding of small GTPases which are therefore a key regulatory element regulating kinase activity of group I family members.

Co-expression of both PAK-4 and Cdc42 results in the translocation of PAK-4 to the golgi and stimulates the induction of filopodia in several cell types. Filopodia formation has been shown to be dependent on both PAK-4 kinases activity and its interaction with Cdc42. A recent report showed that extracellular stimuli like the cytokine HGF can stimulate PAK-4 activity and leads to the phosphorylation of the cytoskeletal regulatory protein LIMK1 and the proapoptotic protein BAD linking PAK-4 to apoptotic pathways. Indeed, activated PAK-4 has been shown to protect cells from apoptosis.

PAK-4 knockout mice are embryonic lethal by embryonic day 11.5, most likely due to a defect in the fetal heart. PAK-4 -/- embryos show neuronal abnormalities. Like neuronal differentiation has been shown to be largely inhibited, axonal outgrowth is impaired, and neurons failed to migrate to their proper locations. In addition, PAK-4 -/- embryos show defects in proper folding of the caudal portion of the neural tube, resulting in the formation of two neural lumens.

It is likely that PAK-4 plays a relevant role in oncogenic transformation. Kinase inactive PAK-4 inhibits focus formation by oncogenic Dbl or oncogenic Ras and elevated expression of PAK-4 has been reported in a high percentage of tumor cells.

We determined therefore the structure of PAK4 in complex with the inhibitor CGP74514A; N-(cis-2-Aminocyclohexyl)-N-(3-chlorophenyl)-9-ethyl-9H-purine-2,6-diamine, which has been originally identified as a strong cdk1 kinase inhibitor. In complex with this inhibitor the entire structure of PAK4 kinase domain was well defined including the phosphorylated activation segment.

In addition, we determined two apo-structures of PAK4 using diffraction data measured on two different crystal forms. Comparison of the two high resolution structures gave interesting insights into conformational flexibility of this kinase.

We also determined the structure of PAK4 in complex with a consensus substrate peptide.

Materials and Methods

Entry Clone Source: TKC

Entry Clone Accession: n/a

SGC Construct ID: PAK4A-c010

GenBank GI number: gi|5031975

Final protein sequence ( PAK4 GST-fusion ):
mspilgywkikglvqptrllleyleekye
ehlyerdegdkwrnkkfelglefpnlpyy
idgdvkltqsmaiiryiadkhnmlggcpk
eraeismlegavldirygvsriayskdfe
tlkvdflsklpemlkmfedrlchktylng
dhvthpdfmlydaldvvlymdpmcldafp
klvcfkkrieaipqidkylksskyiawpl
qgwqatfgggdhppksdlevlfq*gplgs
SPQREPQRVSHEQFRAALQLVVDPGDPRS
YLDNFIKIGEGSTGIVCIATVRSSGKLVA
VKKMDLRKQQRRELLFNEVVIMRDYQHEN
VVEMYNSYLVGDELWVVMEFLEGGALTDI
VTHTRMNEEQIAAVCLAVLQALSVLHAQG
VIHRDIKSDSILLTHDGRVKLSDFGFCAQ
VSKEVPRRKSLVGTPYWMAPELISRLPYG
PEVDIWSLGIMVIEMVDGEPPYFNEPPLK
AMKMIRDNLPPRLKNLHKVSPSLKGFLDR
LLVRDPAQRATAAELLKHPFLAKAGPPAS
VPLMRQNRTR

PreScission(TM) (rhinovirus 3C)- protease cleavable ( * ) GST tag

After cleavage:
gplgs SPQREPQRVSHEQFRAALQLVVDP
GDPRSYLDNFIKIGEGSTGIVCIATVRSS
GKLVAVKKMDLRKQQRRELLFNEVVIMRD
YQHENVVEMYNSYLVGDELWVVMEFLEGG
ALTDIVTHTRMNEEQIAAVCLAVLQALSV
LHAQGVIHRDIKSDSILLTHDGRVKLSDF
GFCAQVSKEVPRRKSLVGTPYWMAPELIS
RLPYGPEVDIWSLGIMVIEMVDGEPPYFN
EPPLKAMKMIRDNLPPRLKNLHKVSPSLK
GFLDRLLVRDPAQRATAAELLKHPFLAKA
GPPASIVPLMRQNRTR

Vector: pGEX-6P2. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Host : BL21 (DE3)

Growth medium, induction protocol: Starter cultures from freshly transformed colonies in 10 ml LB, 0.1 mg/ml ampicillin were grown overnight. This was diluted 1:1000 in fresh media (6L) and was grown at 37°C to an OD₆₀₀ of 0.3 and than transferred to 18°C. Expression was induced at an OD₆₀₀ of 0.8 using 1 mM IPTG. Cells were harvested after 12h by centrifugation, transferred to 50-ml tubes, and frozen in liquid nitrogen.

Extraction buffer, extraction method: Extraction buffer: 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 1mM PMSF. The cell pellets (20 g wet wt) were re-suspended in 50 ml extraction buffer, and lysed by a high pressure cell disrupter. Supernatant was centrifuged for 30 minutes at 60,000 rpm.

Column 1 : Glutathione Sepharose 4B affinity, 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.

Buffers: Binding buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM DTT .

Procedure: Supernatant was applied at gravity flow, followed by a wash with 30 ml binding buffer. The GST-fusion was cleaved while bound to the column by addition of PreScission protease. The column was gently rotated overnight a 4°C. The protein was subsequently eluted with 3 bed volumes of binding buffer.

Column 2 : SEC (S75 or S200)

SEC-Buffers: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM DTT .

Procedure: Fractions containing PAK4 were concentrated and applied to a S75 gel fitration column equilibrated in SEC buffer. PAK4 eluted with a retention time corresponding to a monomeric protein of 40 kDa.

Protein concentration and characterization: Centricons 10 kDa cut off in same buffer. The mass of the recombinant Pak4 corresponded to the expected mass and one PO₄ moiety. The protein was >95% pure as judged by SDS PAGE. ESI-MS spectra showed that the recombinant protein had a molecular weight of 34456- which corresponds to the mono-phosphorylated protein, in agreement with the theoretical mass of 34376 Da

Crystallization:

Pak4 - CGP74514A complex: Pak4 was concentrated in the presence of 1 mM CGP74514A; N-(cis-2-Aminocyclohexyl)-N-(3-chlorophenyl)-9-ethyl-9H-purine-2,6-diamine to a final concentration of 10 mg/ml. The protein was crystallized is 96 well sitting drop Greiner plates using the vapour diffusion method at 4°C mixing 100 nl protein solution with a well solution containing 1.2 M ammonium sulfate, 15% PEG200 and 100 mM Tris pH 8.0. The crystals grew within 3 days and were frozen in a buffer containing the well solution and 25% ethylene glycole.

Pak4 apo hexagonal crystal form: Pak4 was concentrated to 10 mg/ml. The protein was crystallized is 96 well sitting drop Greiner plates using the vapour diffusion method at 4°C mixing 100 nl protein solution with a well solution containing 1.5M NaCl and 10% (v/v) ethanol. Crystals were frozen in the crystallization buffer containing 25% ethylene glycole.

Pak4 apo tetragonal crystal form: Pak4 was concentrated to 10 mg/ml. The protein was crystallized is 96 well sitting drop Greiner plates using the vapour diffusion method at 4°C mixing 100 nl protein solution with a well solution containing 0.20M K 3 (cit); 0.1M BTProp pH 6.5; 20.0% PEG 3350; 10.0% EtGly. Crystals were frozen in the crystallization buffer containing 15% ethylene glycole.

Pak4 substrate complex Pak4 was concentrated to 9.3 mg/ml. The protein was crystallized is 96 well sitting drop Greiner plates using the vapour diffusion method at 4°C mixing 100 nl protein solution with a well solution containing 1.7M ammonium sulfate, 15% PEG400, 0.1M Tris pH 8.0. Crystals were frozen in the crystallization buffer containing 20% ethylene glycole.

Data Collection & Resolution:

74 (2BVA) - 2.3Å ; X-ray source: SLS-X10

74a (2CDZ) - 2.3Å ; X-ray source: Rigaku FRE rotating anode generator

273 (2J0I) - 1.6 Å ; X-ray source: SLS-X10

74b (2QON) 1.75 Å ; X-ray source: SLS-X10

References

Lei M, Robinson MA, Harrison SC. The active conformation of the PAK1 kinase domain. (2005). Structure. 13(5):769-778.

Jaffer ZM, Chernoff J. p21-activated kinases: three more join the Pak. (2002) Int J Biochem Cell Biol., 34:713-717.

Lei M, Lu W, Meng W, Parrini MC, Eck MJ, Mayer BJ, Harrison SC. (2000) Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch. Cell, 102:387-397.

Eswaran, J., Lee, W.H., Debreczeni, J.E., Filippakopoulos, P., Turnbull, A., Fedorov, O., Deacon, S.W., Peterson, J.R., and Knapp, S. (2007). Crystal Structures of the p21-activated kinases PAK4, PAK5, and PAK6 reveal catalytic domain plasticity of active group II PAKs. Structure 15 , 201-213.

Rennefahrt, U.E., Deacon, S.W., Parker, S.A. , Devarajan, K., Beeser, A., Chernoff, J., Knapp, S., Turk, B.E., and Peterson, J.R. (2007). Specificity profiling of Pak kinases allows identification of novel phosphorylation sites. J. Bio. Chem. 282 , 15667-15678.