Human protein kinase C-like 2

Target ID:

PRKCL2A

Aliases:

MGC150606MGC71074Pak-2PAK2PKN2PRK2PRKCL2PRO2042

Deposition Date:

Friday 28th February 2014

Families:

Protein Kinase

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

4CRS

Authors:

Mathea, S., Elkins, J.M., Shrestha, L., Szklarz, M., Tallant, C., Newman, J.A., Cooper, C.D., Shrestha, B., Tumber, A., Cocking, R., Salah, E., von Delft, F., Arrowsmith, C., Edwards, A.M., Bountra, C., Knapp, S.

Site:

Oxford

4CRS

tabs

Structure Details

Protein kinase C-like 2 (PRKCL2), also known as protein kinase N2 (PKN2) or PRK2, is a member of the AGC (protein kinases A, G, C) family of kinases which are characterised by an extended C-terminus which binds back onto the N-terminal lobe of the kinase. PKN2 has an essential role in the cell cycle; knockdown if PKN2 caused a late-stage cytokinesis defect where cells failed to undergo abscission (Schmidt et al., 2007). PKN2 is responsible for phosphorylation of hepatitis C virus RNA polymerase (Kim et al., 2012, 2004) which is important in viral replication. PKN2 supports migration and invasion of 5637 bladder cancer cells (where PKN2 is relatively highly expressed) (Lachmann et al., 2011). We have solved the crystal structure of the kinase domain of PKN2 in complex with ATPγS to 2.75 Å resolution. The structure reveals the typical AGC kinase tail (in red in the figure) bound to the N-terminal lobe of PKN2 via its 'hydrophobic motif'.

Materials and Methods

PRKCL2

PDB:4CRS

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:Origene
SGC Clone Accession:
Tag:N-terminal His6-Z-TEV
Host:Insect cells (Sf9)

Construct

Prelude:
Sequence:MGHHHHHHSSGVDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLLAEAKKLNDAQPKGTENLYFQSMSQQRFQFNLQDFRCCAVLGRGHFGKVLLAEYKNTNEMFAIKALKKGDIVARDEVDSLMCEKRIFETVNSVRHPFLVNLFACFQTKEHVCFVMEYAAGGDLMMHIHTDVFSEPRAVFYAACVVLGLQYLHEHKIVYRDLKLDNLLLDTEGFVKIADFGLCKEGMGYGDRTSTFCGTPEFLAPEVLTETSYTRAVDWWGLGVLIYEMLVGESPFPGDDEEEVFDSIVNDEVRYPRFLSTEAISIMRRLLRRNPERRLGASEKDAEDVKKHPFFRLIDWSALMDKKVKPPFIPTIRGREDVSNFDDEFTSEAPILTPPREPRILSEEEQEMFRDFDYIADWC residues marked in bold are cleaved by TEV protease
Vector:pFB-6HZB

Growth

Medium:
Antibiotics:
Procedure:

Purification

Buffers
Procedure
A 15 g cell pellet was thawed, resuspended in 100 mL lysis buffer (50 mM HEPES/NaOH pH 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP) and sonicated (2 minutes, amplitude 35%, on ice). The cell lysate was cleared by centrifugation (45 minutes, 100,000x g, 4°C). The supernatant was combined with 5 mL Ni Sepharose and loaded onto a gravity flow column. The resin was rinsed thrice with lysis buffer. Proteins bound to the resin were eluted with 4x 12.5 mL elution buffer (50 mM HEPES/NaOH pH 7.4, 500 mM NaCl, 300 mM imidazole, 0.5 mM TCEP) and diluted with 50 mL no salt buffer (20 mM HEPES/NaOH pH 7.4, 0.5 mM TCEP). The next purification step was performed using an AKTAprime system combined with a 5 ml SP Sepharose column. After equilibrating the column with no salt buffer, the sample was loaded. The column was rinsed with 10% high salt buffer (20 mM HEPES/NaOH pH 7.4, 2.5 M NaCl, 0.5 mM TCEP). The protein was eluted by a linear gradient of 10-100% high salt buffer in 100 mL. Fractions containing protein were pooled, combined with recombinant TEV protease (mass ratio 1:25) and incubated with rotation (over night, 4°C). Following this, 5 mL Ni Sepharose was added, and the suspension was loaded onto a gravity flow column. The flowthrough was collected and concentrated to 5.2 mL. Finally, the protein was polished by gel filtration using an AKTAxpress system with an S200 16/600 column and GF buffer (20 mM HEPES/NaOH pH 7.4, 500 mM NaCl, 0.5 mM TCEP). Fractions containing protein were pooled, concentrated and stored at -80°C.

Extraction

Buffers
Procedure
Exponentially growing Sf9 cells (2x 106 cells/mL) were infected with high titre baculovirus stock (1:65) and incubated in shaker flasks (60 hours, 90 rpm, 27°C). Following this, the cell suspensions were centrifuged (15 minutes, 800x g, 4°C), and the cell pellets resuspended in PBS. After another centrifugation (15 minutes, 800x g, 4°C), the cell pellets were stored at -80°C.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals grew from a 1:2 ratio of protein solution (6.7 mg/mL) and precipitant solution (1.7 M (NH4)2SO4, 0.1 M citrate pH 5.8, 0.2 M Na/K tartrate), using the vapour diffusion method.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected by equilibration into precipitant solution containing 25% ethylene glycol and flash frozen in liquid nitrogen. Data was collected at Diamond, beamline I02.
Data Processing:

References

Kim, M.-G., Moon, J.-S., Kim, E.-J., Lee, S.-H., and Oh, J.-W. (2012). Destabilization of PDK1 by Hsp90 inactivation suppresses hepatitis C virus replication through inhibition of PRK2-mediated viral RNA polymerase phosphorylation. Biochem. Biophys. Res. Commun. 421, 112-118.
Kim, S.-J., Kim, J.-H., Kim, Y.-G., Lim, H.-S., and Oh, J.-W. (2004).
Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation. J. Biol. Chem. 279, 50031-50041.
Lachmann, S., Jevons, A., De Rycker, M., Casamassima, A., Radtke, S., Collazos, A., and Parker, P.J. (2011). Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration. PLoS One 6, e21732.
Schmidt, A., Durgan, J., Magalhaes, A., and Hall, A. (2007). Rho GTPases regulate PRK2/PKN2 to control entry into mitosis and exit from cytokinesis. EMBO J. 26, 1624-1636.