Human protein kinase C, eta - C2 domain

Target ID:

PKCH

Aliases:

MGC26269MGC5363nPKC-etaPKC-LPKCLPRKCL

Deposition Date:

Wednesday 4th January 2006

Families:

Protein Kinase

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2FK9

Authors:

J.R.WALKER,D.R.LITTLER,P.J.FINERTY JR.,F.MACKENZIE,E.M.NEWMAN,J.WEIGELT,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2FK9

tabs

Structure Details

Skin is composed of four layers of cells; the top three are each derived by continuous differentiation from its neighboring lower layer.
A permanent extracellular calcium ion concentration gradient determines the differentiated state, with low calcium levels at the basal undifferentiated layer and highest levels at the terminally differentiated cornified (top) layer (Denning, 2004).
In conjunction with other skin specific second messengers, calcium activates a number of signal transduction pathways in keratinocytes, including those containing PKC, PI3K, and TK. Upon calcium activation, basal keratinocytes stop proliferating, begin differentiating, and move toward the surface.

Activation of the serine/threonine kinase prkch is consistently observed during epidermal differentiation,
as suggested by genetic and pharmacological manipulation of prkch activity.
Pkchâ€™s role as differentiating factor is also consistent with the susceptibility to skin cancer as well as other carcinomas that occurs with down-regulation of prkch (Chida et al., 2003; Aeder et al., 2004; Martin and Hussaini, 2005).

Although calcium is not a direct activator of pkch, skin lipids (cholesterol sulfate, cerebroside, and sulfatide) that are formed during squamous cell differentiation are potent activators of the eta isoform of the pkc family (Denning et al., 1995; Kuroki et al., 2000).
And although the phenotype of prkch knockout mice is mild, probably due to isoform compensation, wound healing is impaired (Chida et al., 2003).

To elucidate the mechanism of ligand mediated activation of human prkch, we have determined the high resolution structure of its C2 domain.
In contrast to other C2â€™s, such as the pkc-beta C2, multiple histidines line the putative active site, hinting at a negatively-charged ligand.
This structure represents groundwork for understanding how prkch is activated by skin specific messengers and for the discovery and development of compounds that could have pharmacological power.

Materials and Methods

Prkch C2 domain

PDB:2FK9

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC037268
Entry Clone Source:MGC AT24-D3 pBluescriptR
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated thrombin protease cleavage site: mgsshhhhhhssglvpr*gs(M)
Host:

Construct

Prelude:
Sequence:mgsshhhhhhssglvprlgsMSSGTMKFNGYLRVRIGEAVGLQPTRWSLRHSLFKKGHQLLDPYLTVSVDQVRVGQTSTKQKTNKPTYNEEFCANVTDGGHLELAVFHETPLGYDHFVANCTLQFQELLRTTGASDTFEGWVDLEPEGKVFVVITLTG
Vector:p28-LIC-Thrombin

Growth

Medium:
Antibiotics:
Procedure:The Prkch C2 domain was expressed in E. coli (DE3) in Terrific Broth (TB) in the presence of kanamycin (50 µg/mL). A stab culture was taken from glycerol stocks and inoculated into 100mL of LB with 50ug/mL kanamycin in a 250mL flask and incubated with shaking at 250rpm overnight at 37 ºC. The culture was transfered into 1.8L TB with 50ug/mL kanamycin and 0.6mL of antiforam (Sigma) in 2L bottles and cultured using the LEX system to an OD600 of 4.0-5.0 before induction. The culture was colled to 15ºC and isopropyl-1-thio-D-galactopyranoside (IPTG) was added to 100ug/mL then incubated overnight at 15ºC before harvesting by centrifugation and storage at -80ºC.

Purification

Procedure
4M imidazole was added to the cleared cell lysate to bring it to a final concentration of 5mM imidazole before loading onto 4 mL bed-volume of Ni-NTA agarose affinity resin (Qiagen #30250). The column was then washed with 50mL of binding buffer and 50mL of Wash buffer (50mM Tris, 500mM NaCl, 20mM Imidazole, 0.5mM TCEP) before eluting into 7mL of Elution buffer (50mM Tris, 500mM NaCl, 250mM imidazole, 0.5mM TCEP). Using an AKTAxpress (GE Healthcare) this eluate was loaded onto an An XK 16x65 column packed with HighLoad Superdex 200 resin that was pre-equilibrated in Binding buffer. This gel filatration column was run at a flow rate of 1.5 mL/min and 2mL fractions collected. The Prkch C2 domain eluted with a single major peak, the fractions from this peak were pooled and concentrated to 1.4mg/mL using an Amicon YM10 centrifugal filter device and set into crystal trays.

Extraction

Procedure
The cell-pellet was thawed, then resuspended in 50mL of binding buffer (50mM Tris, 0.5M NaCl, 0.5mM TCEP pH 8.0) containing Sigma's protease inhibitor cocktail (P2714-1BTL). The thawed cells were homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds. The cells were then lysed by sonication (Virtis408912, Virsonic) on ice with a 10 seconds pulse at half-maximal frequency then 10 seconds rest for a total of 5 minutes sonication time. The resulting lysate was then centrifuged at 63 000 xg for 30 minutes at 10 ºC.
Concentration:
Ligand
MassSpec:
Crystallization:The purified Prkch C2 domain was crystallized using the hanging drop vapour diffusion method. On glass slides 3uL of protein at 1.1 mg/mL was mixed with an equal volume of resevoir solution that consisted of 22% w/v polyethylene glycol 3350 and 0.2M CaCl2. The drop was sealed against 1mL of reseveroir and allowed to reach equilibrium at 18 ºC, crystals grew overnight. After harvesting crystal were soaked in a cryo-solution consisting of reservoir plus 300mg/mL D-glucose before freezing in liquid nitrogen and mounting for X-ray diffraction studies.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Aeder,S.E., Martin,P.M., Soh,J.W., and Hussaini,I.M. (2004). PKC-eta mediates glioblastoma cell proliferation through the Akt and mTOR signaling pathways. Oncogene 23, 9062-9069.

Chida,K., Hara,T., Hirai,T., Konishi,C., Nakamura,K., Nakao,K., Aiba,A., Katsuki,M., and Kuroki,T. (2003). Disruption of protein kinase C-eta results in impairment of wound healing and enhancement of tumor formation in mouse skin carcinogenesis. Cancer Research 63, 2404-2408.

Denning,M.F. (2004). Epidermal keratinocytes: regulation of multiple cell phenotypes by multiple protein kinase C isoforms. International Journal of Biochemistry & Cell Biology 36, 1141-1146.

Denning,M.F., Kazanietz,M.G., Blumberg,P.M., and Yuspa,S.H. (1995). Cholesterol Sulfate Activates Multiple Protein-Kinase-C Isoenzymes and Induces Granular-Cell Differentiation in Cultured Murine Keratinocytes. Cell Growth & Differentiation 6, 1619-1626.

Kuroki,T., Ikuta,T., Kashiwagi,M., Kawabe,S., Ohba,M., Huh,N., Mizuno,K., Ohno,S., Yamada,E., and Chida,K. (2000). Cholesterol sulfate, an activator of protein kinase C mediating squamous cell differentiation: a review. Mutation Research-Reviews in Mutation Research 462, 189-195.

Martin,P.M. and Hussaini,I.M. (2005). PKC eta as a therapeutic target in glioblastoma multiforme. Expert Opinion on Therapeutic Targets 9, 299-313.