PKMz - Revision history

Monakhos at 15:33, 1 May 2012

2012-05-01T15:33:40Z

← Older revision		Revision as of 08:33, 1 May 2012
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	=== Protein Kinase M-zeta (PKMz) & Zeta Inhibitory Peptide ===		=== 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		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

Monakhos at 15:33, 1 May 2012

2012-05-01T15:33:28Z

← Older revision		Revision as of 08:33, 1 May 2012
Line 7:		Line 7:


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

	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		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

Monakhos at 23:03, 20 April 2012

2012-04-20T23:03:57Z

@@ Line 1: / Line 1: @@
 === Protein Kinase M-zeta (PKMz)===
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 :- constitutively phosphorylates all PKC isoforms
 :- required for most downstream kinase activity

Monakhos at 22:46, 20 April 2012

2012-04-20T22:46:16Z

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+----
 === Methods ===
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 *Ser(P) PKC substrate antibody
 *Phospho-MARK2 antibody
-*DsRed antibody (recognizes mRFP and tdTomato))
+*DsRed antibody (recognizes mRFP and tdTomato)
 *Beta-actin antibody
 *pAL antibody (recognizes phosphorylated activation loop of PKC isozymes)
@@ Line 91: / Line 92: @@
 *reaction aliquots were spotted onto chromatography paper, washed, and put into scintillation buffer
 *radioactivity was then determined by liquid scintillation counting

Monakhos at 21:38, 20 April 2012

2012-04-20T21:38:13Z

@@ Line 19: / Line 19: @@
 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.
@@ Line 78: / Line 82: @@
 *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

Monakhos at 21:19, 20 April 2012

2012-04-20T21:19:18Z

← Older revision		Revision as of 14:19, 20 April 2012
Line 14:		Line 14:
	:- constitutively phosphorylates all PKC isoforms		:- constitutively phosphorylates all PKC isoforms
	:- required for most downstream kinase activity		:- required for most downstream kinase activity


			== 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.


			;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

Monakhos at 20:28, 20 April 2012

2012-04-20T20:28:18Z

← Older revision		Revision as of 13:28, 20 April 2012
Line 1:		Line 1:
	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 ~~putative~~ 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.
			=== Protein Kinase M-zeta (PKMz)===

			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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2012-04-20T20:18:11Z

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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 putative 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.