PKMz

Experiment to Test if ZIP blocks PKMz

Wu-Zhang, Schramm, Nabavi, Malinow, Newton (2012). Cellular Pharmacology of Protein Kinase Mzeta (PKMzeta) Contrasts with its In Vitro Profile. The Journal of Biological Chemistry.

Link to the Journal Article

Protein Kinase M-zeta (PKMz) & Zeta Inhibitory Peptide (ZIP)

This guide covers the putative role of protein kinase M-zeta (PKMz) in synaptic plasticity and long term memory maintenance. Evidence that PKMz is required for LTP and for the maintenance of several forms of memory has depended almost exclusively on the use of pharmacological approaches that use a zeta inhibitory peptide (ZIP). ZIP is a myristoylated PKCz-inhibiting peptide derived from the autoinhibitory pseudosubstrate peptide sequence within PKCz, chelerythrine, an apoptosis-inducing compound that is marketed, and extensively used, as a PKC inhibitor, and staurosporine, a general protein kinase inhibitor

• Zeta Inhibitory Peptide

- myristoylated form of chelerythrine

• Chelerythrine

- autoinhibitory substrate for PKCz

• Staurosporine

- general protein kinase inhibitor

- inhibits phosphoinositide-dependent kinase-1 (PDK1)

• PDK1

- constitutively phosphorylates all PKC isoforms

- required for most downstream kinase activity

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Methods

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Materials

• ZIP
• scrambled ZIP
• chelerythrine
• staurosporine
• bisIV
• Ser(P) PKC substrate antibody
• Phospho-MARK2 antibody
• DsRed antibody (recognizes mRFP and tdTomato)
• Beta-actin antibody
• pAL antibody (recognizes phosphorylated activation loop of PKC isozymes)

Plasmids

• CKAR (C kinase activity reporter construct)
• PKMz+RFP tag vector
• mRFP vector control
• tdTomato vector control
• Sindbis virus was packaged with vectors

Cell Culture and Transfection

• HeLa and 293T and COS-7 cells were maintained in DMEM
• Transfections were carried out using FuGENE 6 or jetPRIME

Brain Slices and Infection

• hippocampal brain slice cultures from PD6 rat pups
• injected with Sindbis virus containing either tdTomato or PKMz+RFP
• virus allowed to express for 24h
• two brain sections per group were homogenized together
• protein concentrations were determined (using BCA) for Western blotting

Immunoblotting

• the 293T and HeLa cells were used for immunoblotting
• transfected with either mRFP or PKMz+RFP
• plated in wells and grown to confluence then lysed
• lysates were separated using SDS-polyacrylamine gels
• separated lysates were transferred to PVDF membranes and probed with antibody

Immunoprecipitation

• the 293T cells were used for immunoprecipitation
• transfected with either mRFP or PKMz+RFP
• plated in wells and grown to confluence then lysed
• DsRed antibody (used to detect RFP) was added to lysates
• samples were then analyzed by SDS-PAGE and immunoblotting (described above)

Live Cell Fluorescence Imaging

• the COS-7 cells were used for fluorescence imaging
• COS-7 cells were transfected with CKAR & mRFP or CKAR & PKMz+RFP
• FRET images were collected every 15 s via MicroMax camera
• time-lapes images of cyan or yellow fluorescent protein (CFP)(YFP) were also captured
• for each manually selected cellular region a CFP/FRET ratio was calculated
• FRET ratios were combined from ~11 cells per group over 3 independent experiments

In Vitro Kinase Activity Assay

• effect of 1 uM ZIP or scrambled ZIP on PKMz activity was assayed in vitro
• the incorporation of radioactive phosphate from [³²P]ATP into synthetic PKC-selective substrate peptide was monitored
• purified PKMz was used
• enzymatic reaction took place in HEPES buffer for 11 min
• reaction aliquots were spotted onto chromatography paper, washed, and put into scintillation buffer
• radioactivity was then determined by liquid scintillation counting

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Results

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For the first immunoblotting experiment used 293T cells, which I'm guessing are transformed HEK-293 cells which have origins as human embryonic kidney cells. NB - they are not neurons. No big deal though, they found that PKMz enhanced the phosphorylation of multiple substrates.

The antibody used for blots was a Ser(P) PKC substrate antibody that detects any substrates whose phosphorylation is enhanced by PKMz. They compared 293T cells that overexpressed PKMz+RFP with cells expressing RFP alone using Ser(P) and found that PKMz robustly enhanced the phosphorylation of multiple PKC substrates (in particular a protein with a molecular mass of ~180 kDa).

A subset of 293T cells were treated with ZIP, scrambled ZIP, or chelerythrine; none of which altered the phosphorylation activity of PKMz. This was evidenced by the fact that there was the same amount of 180 kDa mystery protein detected by Ser(P) across the three groups.

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