Human p21 activated kinase 5

Target ID:

PAK5A

Aliases:

KIAA1264MGC26232PAK5

Deposition Date:

Friday 25th November 2005

Families:

Non-protein Kinase

Related Diseases:

CancerSignallingNeurobiology

Structure type:

apo

Download Coordinates:

2F57

Authors:

J.ESWARAN,A.TURNBULL,E.UGOCHUKWU,E.PAPAGRIGORIOU,J.BRAY,S.DAS,P.SAVITSKY,C.SMEE,N.BURGESS,O.FEDOROV,P.FILIPPAKOPOULOS,F.VON DELFT,C.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,A.EDWARDS,S.KNAPP,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2F57

tabs

Structure Details

The p21-activated protein kinases (PAKs) are serine/threonine protein kinases of the STE20 family which are involved in Cdc42 and Rac1 signaling.
PAK kinases contain a well defined p21-binding domain (PBD) including the CRIB motif.
Six members of this kinase family have been identified in human.
The PAK kinase family is subdivided into two groups according to kinase domain homology and the existence of a conserved autoinhibitory domain (AID). Group I PAKs (PAKs 1, 2, and 3) exhibit 80 to 90% sequence identity within their catalytic domains and contain a AID domain, whereas the recently identified group II PAKs (PAK 4, 5, and 6), share only about 40 to 50% identity to group I kinase domains and do not contain obvious AID domains.
Thus, the mechanisms that regulate group II PAKs are unclear, but the absence of a conserved AID indicates that the modes of regulation differ significantly from group I PAKs.

PAK5 is highly expressed in the brain and neuronal tissues and is expressed at lower levels in several other tissues such as liver, kidney and heart.
PAK5, unlike PAK1, cannot complement an STE 20 mutation in Saccharomyces cerevisiae.
PAK5 has been shown to bind GTPases Cdc42 and Rac, but these GTPases do not regulate PAK5 kinase activity, which is constitutively active and has a higher specific activity than any other PAK family member.
PAK5 is localized to mitochondria, and localization is independent of kinase activity or Cdc42 binding.

Knockout mice of PAK5 show no apparent phenotype potentially due to functional redundancy with other PAKs.
However, PAK5 has been shown to be an important mediator in the signaling pathway by which Rho GTPases control cytoskeletal changes necessary for promoting neurite outgrowth.
Similar to other Group II PAKs, PAK5 is activated by MKK6, consistent with the presence of a conserved TPY motif in the activation domain.
PAK5 prevents apoptosis induced by camptothecin and C2-ceramide by phosphorylating BAD ( BCL 2-antagonist of cell death) on Ser-112 in a protein kinase A-independent manner and prevents the localization of BAD to mitochondria, thereby inhibiting the apoptotic cascade.
PAK5 has also been to keep microtubules stable through down regulation of MARK2 but ir destabilizes the F-actin network so that stress fibers and focal adhesions disappear and cells develop filopodia.

Materials and Methods

PAK5

PDB:2F57

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|24308191
Entry Clone Source:MGC
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m)TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smSRVSHEQFRAALQLVVSPGDPREYLANFIKIGEGSTGIVCIATEKHTGKQVAVKKMDLRKQQRRELLFNEVVIMRDYHHDNVVDMYSSYLVGDELWVVMEFLEGGALTDIVTHTRMNEEQIATVCLSVLRALSYLHNQGVIHRDIKSDSILLTSDGRIKLSDFGFCAQVSKEVPKRKXLVGTPYWMAPEVISRLPYGTEVDIWSLGIMVIEMIDGEPPYFNEPPLQAMRRIRDSLPPRVKDLHKVSSVLRGFLDLMLVREPSQRATAQELLGHPFLKLAGPPSCIVPLMRQ
Vector:pLIC-SGC1

Growth

Medium:
Antibiotics:
Procedure:1mL from a 10 mL LB overnight culture containing 50 µg/mL kanamycin was used to inoculate 1 liter of LB media containing 50 µg/mL kanamycin. Cultures were grown at 37°C until the OD600 reached ~0.3. After that the temperature was adjusted to 18°C. Expression was induced for 4 hours using 1mM IPTG at an OD600 of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol.

Purification

Procedure
Column 1: Ni-affinity chromatography.

Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500mM NaCl, 5% Glycerol. Wash buffer: 50 mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole, 5% glycerol. Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 50 to 250mM Imidazole, 5% Glycerol.

Procedure: 5 mL of 50% Ni-NTA slurry (Qiagen) was applied to a 1.5 x 10 cm gravity column. The column was equilibrated with 30 mL binding buffer. The lysate was applied to the column which was subsequently washed with 3 x 10 mL wash buffer. PAK5 was eluted by a step gradient generated by 5 mL portions of elution buffer with increasing concentration of imidazole (concentrations used: 50mM, 100mM, 250mM). The eluted protein fractions were collected and analyzed by SDS - PAGE . Most of PAK5 eluted at an imidazole concentration of 100 mM. After elution DTT was added to a final concentration of 10mM.

Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)

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

Procedure: The fractions eluted of the Ni-affinity chromatography were pooled and concentrated to about 1 mL using Centricon concentrators (10kDa cut off). The concentrated protein was applied to a Superdex S75 column equilibrated in SEC buffer at a flow rate of 1 mL/min. PAK5 eluted at 65 minutes corresponding to a retention time of a monomeric protein of that size. Eluted fractions were 95% pure as judged by SDS - PAGE .

Protein concentration: Centricon with a 10kDa cut off in SEC -buffer

Extraction

Procedure
Cell pellets were lysed using a high pressure cell disrupter. The lysate was centrifuged at 50,000 rpm for 40 minutes and the supernatant collected for purification.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/mL by mixing 100nl of the concentrated protein with 100nl of a well solution containing 0.20M Na/KPO4, 0.1M Bis Tris Propane pH 7.5, 20.0% PEG 3350, 10.0% Etylene glycole at 4°C.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Dan, C., Nath, N., Liberto, M., and Minden , A. (2002). PAK5, a new brain-specific kinase, promotes neurite outgrowth in N1E-115 cells. Mol. Cell. Biol. 22 , 567-577.

Cotteret S, Jaffer ZM, Beeser A, Chernoff J. (2003) p21-Activated kinase 5 (Pak5) localizes to mitochondria and inhibits apoptosis by phosphorylating BAD. Mol Cell Biol. 23, 5526-39.

Ching YP, Leong VY, Wong CM, Kung HF. (2003) Identification of an autoinhibitory domain of p21-activated protein kinase 5. J Biol Chem. 278, 33621-4.

Matenia D, Griesshaber B, Li XY, Thiessen A, Johne C, Jiao J, Mandelkow E, Mandelkow EM. (2005) PAK5 kinase is an inhibitor of MARK/Par-1, which leads to stable microtubules and dynamic actin. Mol Biol Cell. 16, 4410-22.

Kaur, R., Liu, X., Gjoerup, O., Zhang, A., Yuan, X., Balk, S., Schneider, S., and Lu, M. (2005). Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase. J. Biol. Chem. 280 , 3323-3330.