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Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation

Göransson, Olga LU ; Deak, Maria ; Wullschleger, Stephan ; Morrice, Nick A. ; Prescott, Alan R. and Alessi, Dario R. (2006) In Journal of Cell Science 119(19). p.4059-4070
Abstract
Members of the PAR-1/MARK kinase family play critical roles in polarity and cell cycle control and are regulated by 14-3-3 scaffolding proteins, as well as the LKB1 tumour suppressor kinase and atypical protein kinase C (PKC). In this study, we initially investigated the mechanism underlying the interaction of mammalian MARK3 with 14-3-3. We demonstrate that 14-3-3 binding to MARK3 is dependent on phosphorylation, and necessitates the phosphate-binding pocket of 14-3-3. We found that interaction with 14-3-3 was not mediated by the previously characterised MARK3 phosphorylation sites, which led us to identify 15 novel sites of phosphorylation. Single point mutation of these sites, as well as the previously identified LKB1- (T211) and the... (More)
Members of the PAR-1/MARK kinase family play critical roles in polarity and cell cycle control and are regulated by 14-3-3 scaffolding proteins, as well as the LKB1 tumour suppressor kinase and atypical protein kinase C (PKC). In this study, we initially investigated the mechanism underlying the interaction of mammalian MARK3 with 14-3-3. We demonstrate that 14-3-3 binding to MARK3 is dependent on phosphorylation, and necessitates the phosphate-binding pocket of 14-3-3. We found that interaction with 14-3-3 was not mediated by the previously characterised MARK3 phosphorylation sites, which led us to identify 15 novel sites of phosphorylation. Single point mutation of these sites, as well as the previously identified LKB1- (T211) and the atypical PKC sites (T564/S619), did not disrupt 14-3-3 binding. However, a mutant in which all 17 phosphorylation sites had been converted to alanine residues ( termed 17A-MARK3), was no longer able to bind 14-3-3. Wild-type MARK3 was present in both the cytoplasm and plasma membrane, whereas the 17A-MARK3 mutant was strikingly localised at the plasma membrane. We provide data indicating that the membrane localisation of MARK3 required a highly conserved C-terminal domain, which has been termed kinase-associated domain-1 (KA-1). We also show that dissociation of 14-3-3 from MARK3 did not affect catalytic activity, and that a MARK3 mutant, which could not interact with 14-3-3, was normally active. Finally, we establish that there are significant differences in the subcellular localisation of MARK isoforms, as well as in the impact that atypical PKC overexpression has on 14-3-3 binding and localisation. Collectively, these results indicate that 14-3-3 binding to MARK isoforms is mediated by multiple phosphorylation sites, and serves to anchor MARK isoforms in the cytoplasm. (Less)
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author
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
phosphorylation site, cell polarity, 14-3-3, PAR-1/MARK
in
Journal of Cell Science
volume
119
issue
19
pages
4059 - 4070
publisher
The Company of Biologists Ltd
external identifiers
  • wos:000240630300016
  • scopus:33750457625
  • pmid:16968750
ISSN
0021-9533
DOI
10.1242/jcs.03097
language
English
LU publication?
yes
id
b07cc9da-e90d-44eb-9302-0eb803f561a3 (old id 393064)
date added to LUP
2016-04-01 12:25:37
date last changed
2020-12-08 03:01:37
@article{b07cc9da-e90d-44eb-9302-0eb803f561a3,
  abstract     = {Members of the PAR-1/MARK kinase family play critical roles in polarity and cell cycle control and are regulated by 14-3-3 scaffolding proteins, as well as the LKB1 tumour suppressor kinase and atypical protein kinase C (PKC). In this study, we initially investigated the mechanism underlying the interaction of mammalian MARK3 with 14-3-3. We demonstrate that 14-3-3 binding to MARK3 is dependent on phosphorylation, and necessitates the phosphate-binding pocket of 14-3-3. We found that interaction with 14-3-3 was not mediated by the previously characterised MARK3 phosphorylation sites, which led us to identify 15 novel sites of phosphorylation. Single point mutation of these sites, as well as the previously identified LKB1- (T211) and the atypical PKC sites (T564/S619), did not disrupt 14-3-3 binding. However, a mutant in which all 17 phosphorylation sites had been converted to alanine residues ( termed 17A-MARK3), was no longer able to bind 14-3-3. Wild-type MARK3 was present in both the cytoplasm and plasma membrane, whereas the 17A-MARK3 mutant was strikingly localised at the plasma membrane. We provide data indicating that the membrane localisation of MARK3 required a highly conserved C-terminal domain, which has been termed kinase-associated domain-1 (KA-1). We also show that dissociation of 14-3-3 from MARK3 did not affect catalytic activity, and that a MARK3 mutant, which could not interact with 14-3-3, was normally active. Finally, we establish that there are significant differences in the subcellular localisation of MARK isoforms, as well as in the impact that atypical PKC overexpression has on 14-3-3 binding and localisation. Collectively, these results indicate that 14-3-3 binding to MARK isoforms is mediated by multiple phosphorylation sites, and serves to anchor MARK isoforms in the cytoplasm.},
  author       = {Göransson, Olga and Deak, Maria and Wullschleger, Stephan and Morrice, Nick A. and Prescott, Alan R. and Alessi, Dario R.},
  issn         = {0021-9533},
  language     = {eng},
  number       = {19},
  pages        = {4059--4070},
  publisher    = {The Company of Biologists Ltd},
  series       = {Journal of Cell Science},
  title        = {Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation},
  url          = {http://dx.doi.org/10.1242/jcs.03097},
  doi          = {10.1242/jcs.03097},
  volume       = {119},
  year         = {2006},
}