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.
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.
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.
| Original language | English |
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| Qualification | Doctor |
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| Award date | 2007 Jun 8 |
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| ISBN (Print) | 978-91-85559-83-1 |
| Publication status | Published - 2007 |
Bibliographical note
Defence detailsDate: 2007-06-08
Time: 13:00
Place: Main Lecture Hall, Pathology Building, University Hospital MAS, Malmö
External reviewer(s)
Name: Johansson, Staffan
Title: Professor
Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden
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<div class="article_info">Helena Stensman, Arathi Raghunath and Christer Larsson. <span class="article_issue_date">2004</span>. <span class="article_title">Autophosphorylation suppresses whereas kinase inhibition augments the translocation of protein kinase C? in response to diacylglycerol</span> <span class="journal_series_title">Biol. Chem</span>, <span class="journal_volume">vol 279</span> <span class="journal_pages">pp 40576-40583</span>.</div>
<div class="article_info">Helena Stensman and Christer Larsson. <span class="article_issue_date"></span>. <span class="article_title">Identification of acidic amino acid residues in the PKC? V5 domain that contribute to its sensitivity to diacylglycerol</span> (submitted)</div>
<div class="article_info">Helena Stensman and Christer Larsson. <span class="article_issue_date"></span>. <span class="article_title">Protein kinase C? is important for migration of neuroblastoma cells</span> (manuscript)</div>
Subject classification (UKÄ)
- Clinical Medicine
Free keywords
- Cytology
- oncology
- cancerology
- Cytologi
- Medicin (människa och djur)
- Medicine (human and vertebrates)
- neuroblastoma
- cell migration
- intramolecular interactions
- autophosphorylation
- Protein kinase C
- onkologi
- cancer
