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

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.

14‐3‐3 proteins mediate inhibitory effects of cAMP on salt‐inducible kinases (SIKs)

“…In addition to the SIKs, the MAP/microtubule affinity-regulating kinases (MARKs) subfamily of AMPK related Ser/Thr kinases has also been shown to interact with 14-3-3 proteins [32,44]. Although MARKs have been primarily linked to the regulation of cell polarity, they can also phosphorylate CRTCs and class IIA HDACs [13,14,45,46].…”

Polarity-Regulating Kinase Partitioning-Defective 1/Microtubule Affinity-Regulating Kinase 2 Negatively Regulates Development of Dendrites on Hippocampal Neurons

“…Previously, there appeared another report that one of the family kinases MARK3 is also phosphorylated by aPKC not only on T595 but also on several other residues, which collectively contribute to the regulation of Par1b/MARK2 localization (Goransson et al, 2006). However, there is a common feature in these reports that the localization of Par1b/MARK2 is regulated by phosphorylation from the membrane to the cytosol.…”

A Robust Protocol to Map Binding Sites of the 14-3-3 Interactome: Cdc25C Requires Phosphorylation of Both S216 and S263 to bind 14-3-3

“…For proteins with numerous 14-3-3 binding sites such as MARK2, the peptide array/overlay method can be used to parse out the contributing sites (supplementary Fig. S5) whereas mutational analysis of the complete protein is much more complex and uninformative (29). We also showed that in silico prediction can aid in validating 14-3-3 targets containing at least two binding sequences (supplementary Fig.…”

“…S5). The mammalian MARK3 kinase has been extensively mapped for 14-3-3 binding sites, but no key "gatekeeper site" was found (29). Likely MARK2, similar to MARK3, contains multiple 14-3-3 binding sites; of the known phosphorylation sites on MARK3 (29), 5/7 were positive for 14-3-3 binding by peptide overlay.…”

“…Likely MARK2, similar to MARK3, contains multiple 14-3-3 binding sites; of the known phosphorylation sites on MARK3 (29), 5/7 were positive for 14-3-3 binding by peptide overlay. A study has shown singly substituted Ser/Thr to Ala MARK3 mutants behave as wild type, and only when all 17 candidate Ser/Thr mutations was 14-3-3 binding abolished (29). The PKC-like site at S657 is therefore one of several 14-3-3 binding sites.…”