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Regulation of PKCalpha and the role of PKC in neuroblastoma cell migration

Stensman, Helena LU (2007)
Abstract (Swedish)
Popular Abstract in Swedish

Protein kinas C (PKC) är en familj serin/treonin kinaser som har en viktig roll i regleringen av olika funktioner i cellen. PKC kan delas in i klassiska (alpha, betaI, betaII and gamma), nya (delta, epsilon, eta and theta) och atypiska (zeta and iota/lambda) isoformer beroende på skillnader i hur de regleras. När PKCalpha är inaktivt bidrar intramolekylära bindningar till att proteinet hålls i en stängd form. Vid aktivering kan PKC binda andra proteiner, membran och olika aktivatorer och då bryts de här interaktionerna och PKC får en mer öppen form. Vi har tidigare sett att stimulering med carbachol ger en transient translokering av PKCalpha till plasma membranet. Translokeringen induceras av... (More)
Popular Abstract in Swedish

Protein kinas C (PKC) är en familj serin/treonin kinaser som har en viktig roll i regleringen av olika funktioner i cellen. PKC kan delas in i klassiska (alpha, betaI, betaII and gamma), nya (delta, epsilon, eta and theta) och atypiska (zeta and iota/lambda) isoformer beroende på skillnader i hur de regleras. När PKCalpha är inaktivt bidrar intramolekylära bindningar till att proteinet hålls i en stängd form. Vid aktivering kan PKC binda andra proteiner, membran och olika aktivatorer och då bryts de här interaktionerna och PKC får en mer öppen form. Vi har tidigare sett att stimulering med carbachol ger en transient translokering av PKCalpha till plasma membranet. Translokeringen induceras av ökningar i Ca2+ och PKCalpha kan inte svara på diacylglycerol.



I det här arbetet har vi funnit att autofosforylering av två platser i V5-domänen av PKCalpha gör att proteinet inte kan svara på diacylglycerol och det beror förmodligen på att den diacylglycerol-bindande C1a-domänen är gömd. Vi har identifierat aminosyror i V5-domänen som gör PKCalpha känsligt för diacylglycerol när de är muterade till alanin eller lysin. Samma effekt uppnåddes när fyra lysin i ett lysin-rikt område i C2-domänen muterades till glutamat. Vi tror att de här strukturerna deltar i en intramolekylär binding som döljer den diacylglycerol-bindande C1a-domänen. När den här bindningen bryts blottas C1a-domänen och PKCalpha blir känsligt för diacylglycerol.



Cellers förmåga att migrera är viktig för tumörcellers malignitet. Vi har funnit att aktivering av PKC med 12-O-tetradecanoylphorbol-13-acetate (TPA) ger migration av neuroblastomcellerna SK-N-BE(2)C. Nedreglering av PKCepsilon med siRNA minskar både basal och TPA-inducerad migration och det visar på en viktig roll vid migration för den här isoformen. Varken Erk-signalvägen eller myristoylated alanine-rich C kinase substrate (MARCKS) är viktiga för PKCepsilon-medierad migration även om de kan vara involverade i TPA-inducerad migration. (Less)
Abstract
Members of the protein kinase C (PKC) family of serine/threonine kinases play critical roles in cellular regulation. Depending on differences in regulation, PKCs can be divided into classical (alpha, betaI, betaII and gamma), novel (delta, epsilon, eta and theta) and atypical (zeta and iota/lambda) isoforms. When PKCalpha is inactive it is maintained in a closed conformation by intramolecular interactions. Upon activation these interactions are disrupted by binding proteins, membranes and activators.



We have previously seen that stimulation with carbachol induces a transient translocation of PKCalpha to the plasma membrane. This is induced by an increase in Ca2+ and PKCalpha does not respond to diacylglycerol.

... (More)
Members of the protein kinase C (PKC) family of serine/threonine kinases play critical roles in cellular regulation. Depending on differences in regulation, PKCs can be divided into classical (alpha, betaI, betaII and gamma), novel (delta, epsilon, eta and theta) and atypical (zeta and iota/lambda) isoforms. When PKCalpha is inactive it is maintained in a closed conformation by intramolecular interactions. Upon activation these interactions are disrupted by binding proteins, membranes and activators.



We have previously seen that stimulation with carbachol induces a transient translocation of PKCalpha to the plasma membrane. This is induced by an increase in Ca2+ and PKCalpha does not respond to diacylglycerol.



In this work we have found that autophosphorylation of two sites in the V5 domain of PKCalpha keeps the protein insensitive to diacylglycerol, presumably due to masking of the diacylglycerol-binding C1a domain. We have identified acidic amino acids in the V5 domain that, when mutated to alanines or lysines, renders PKCalpha sensitive to diacylglycerol. Furthermore, mutation of four lysines to glutamate in a lysine-rich cluster in the C2 domain gives a similar effect. We propose that these structures participate in an intramolecular interaction that keeps PKCalpha in a closed conformation hiding the diacylglycerol-binding C1a domain. When disrupting this interaction the C1a domain is exposed and PKCalpha becomes sensitive to diacylglycerol.



The capacity of cells to migrate is crucial for the malignancy of tumour cells. We have found that activation of PKC with 12-O-tetradecanoylphorbol-13-acetate (TPA) induces migration of SK-N-BE(2)C neuroblastoma cells. Downregulation of PKCepsilon with siRNA suppresses both basal and TPA-induced migration indicating an important role for this isoform in migration. Neither the Erk pathway nor myristoylated alanine-rich C kinase substrate (MARCKS) is critical downstream targets of PKCepsilon, but might be involved in TPA-induced migration. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Professor Johansson, Staffan, Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Cytology, oncology, cancerology, Cytologi, Medicin (människa och djur), Medicine (human and vertebrates), neuroblastoma, cell migration, intramolecular interactions, autophosphorylation, Protein kinase C, onkologi, cancer
publisher
Department of Laboratory Medicine, Lund University
defense location
Main Lecture Hall, Pathology Building, University Hospital MAS, Malmö
defense date
2007-06-08 13:00
ISSN
1652-8220
ISBN
978-91-85559-83-1
language
English
LU publication?
yes
id
b2a2690d-014e-482d-99f6-a27c32be0eea (old id 548806)
date added to LUP
2007-09-12 09:54:15
date last changed
2016-09-19 08:44:53
@phdthesis{b2a2690d-014e-482d-99f6-a27c32be0eea,
  abstract     = {Members of the protein kinase C (PKC) family of serine/threonine kinases play critical roles in cellular regulation. Depending on differences in regulation, PKCs can be divided into classical (alpha, betaI, betaII and gamma), novel (delta, epsilon, eta and theta) and atypical (zeta and iota/lambda) isoforms. When PKCalpha is inactive it is maintained in a closed conformation by intramolecular interactions. Upon activation these interactions are disrupted by binding proteins, membranes and activators.<br/><br>
<br/><br>
We have previously seen that stimulation with carbachol induces a transient translocation of PKCalpha to the plasma membrane. This is induced by an increase in Ca2+ and PKCalpha does not respond to diacylglycerol.<br/><br>
<br/><br>
In this work we have found that autophosphorylation of two sites in the V5 domain of PKCalpha keeps the protein insensitive to diacylglycerol, presumably due to masking of the diacylglycerol-binding C1a domain. We have identified acidic amino acids in the V5 domain that, when mutated to alanines or lysines, renders PKCalpha sensitive to diacylglycerol. Furthermore, mutation of four lysines to glutamate in a lysine-rich cluster in the C2 domain gives a similar effect. We propose that these structures participate in an intramolecular interaction that keeps PKCalpha in a closed conformation hiding the diacylglycerol-binding C1a domain. When disrupting this interaction the C1a domain is exposed and PKCalpha becomes sensitive to diacylglycerol.<br/><br>
<br/><br>
The capacity of cells to migrate is crucial for the malignancy of tumour cells. We have found that activation of PKC with 12-O-tetradecanoylphorbol-13-acetate (TPA) induces migration of SK-N-BE(2)C neuroblastoma cells. Downregulation of PKCepsilon with siRNA suppresses both basal and TPA-induced migration indicating an important role for this isoform in migration. Neither the Erk pathway nor myristoylated alanine-rich C kinase substrate (MARCKS) is critical downstream targets of PKCepsilon, but might be involved in TPA-induced migration.},
  author       = {Stensman, Helena},
  isbn         = {978-91-85559-83-1},
  issn         = {1652-8220},
  keyword      = {Cytology,oncology,cancerology,Cytologi,Medicin (människa och djur),Medicine (human and vertebrates),neuroblastoma,cell migration,intramolecular interactions,autophosphorylation,Protein kinase C,onkologi,cancer},
  language     = {eng},
  publisher    = {Department of Laboratory Medicine, Lund University},
  school       = {Lund University},
  title        = {Regulation of PKCalpha and the role of PKC in neuroblastoma cell migration},
  year         = {2007},
}