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

Olga Gransson ) 1 2 Maria Deak 1 Stephan Wullschleger 1 Nick A. Morrice 1 Alan R. Prescott 0 Dario R. Alessi 1 0 University of Dundee, Division of Cell Biology and Immunology , MSI/WTB complex, Dow Street, Dundee, DD1 5EH, Scotland, UK 1 University of Dundee, MRC Protein Phosphorylation Unit, James Black Centre 2 Present address: Lund University , BMC, C11, S-22184 Lund, Sweden - 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 e is dependent on phosphorylation, and necessitates the c phosphate-binding pocket of 14-3-3. We found that n ie interaction with 14-3-3 was not mediated by the previously c characterised MARK3 phosphorylation sites, which led us llS to identify 15 novel sites of phosphorylation. Single point e mutation of these sites, as well as the previously identified fC LKB1- (T211) and the atypical PKC sites (T564/S619), did o not disrupt 14-3-3 binding. However, a mutant in which all la 17 phosphorylation sites had been converted to alanine rn residues (termed 17A-MARK3), was no longer able to bind uo 14-3-3. Wild-type MARK3 was present in both the J cytoplasm and plasma membrane, whereas the 17A MARK3 mutant was strikingly localised at the plasma Introduction Members of the PAR-1/MARK (partition-defective or microtubule-affinity regulating) kinase family are conserved from yeast to humans, and have been shown to play crucial roles in cellular functions such as polarity and cell-cycle control (reviewed in Drewes, 2004; Tassan and Le Goff, 2004). PAR-1 was first isolated in C. elegans, as one of six Par genes required for the formation of anterior-posterior asymetry of the nematode embryo (Guo and Kemphues, 1995; Kemphues et al., 1988; Pellettieri and Seydoux, 2002). PAR-1 homologues have subsequently been identified and studied in a number of organisms, including yeast, Drosophila and mammals (Drewes et al., 1997; Elbert et al., 2005; La Carbona et al., 2004; Shulman et al., 2000; Trinczek et al., 2004). These studies have further implicated a role for PAR-1 in regulating cell polarity and other cellular functions, such as mitogenic signalling and cell-cycle control. Human PAR-1 is encoded by four genes, giving rise to the isoforms MARK1 (PAR-1c), MARK2 (PAR-1b/EMK), MARK3 (PAR-1a/p78/C-TAK1) and MARK4 (PAR1d/MARKL1) (Tassan and Le Goff, 2004). As in C. elegans and Drosophila, human MARK (hMARK) isoforms are membrane. We provide data indicating that the membrane localisation of MARK3 required a highly conserved Cterminal 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. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/19/4059/DC1 asymmetrically localised in epithelial cells (Bohm et al., 1997). Furthermore, MARK1 and MARK2 was required for normal polarisation of kidney- (MDCK cells) or liver epithelium (Bohm et al., 1997; Cohen et al., 2004), and for neurite outgrowth (Biernat et al., 2002; Brown et al., 1999). Expression of MARK4 was upregulated in glioblastomas, as well as in hepatocellular carcinomas, suggesting a role for MARK4 in tumorigenesis (Beghini et al., 2003; Kato et al., 2001). Furthermore, MARK4 expression was also induced during focal cerebral ischemia, and cell viability of neuronal cells was decreased following the overexpression of MARK4 (Schneider et al., 2004). Mammalian PAR-1 was first purified from brain, and named microtubule affinity regulating kinase (MARK), based on its ability to phosphorylate microtubule associated proteins (MAPs) such as MAP2, MAP4 and tau, resulting in their dissociation from microtubules (Drewes et al., 1997; Trinczek et al., 2004). The residue in tau phosphorylated by MARK isoforms, S262, is hyperphosphorylated in Alzheimers disease. Genetic analysis in Drosophila indicated that phosphorylation of this residue primes the hyperphosphorylation of tau by other kinases (Nishimura et al., 2004). 14-3-3 binding to MARK isoforms is mediated, and how it controls MARK activity and localisation, is not yet understood. This could provide an important clue to how MARK function is regulated and is the focus of this study. Results Previously characterised phosphorylation sites are not required for binding of MARK3 to 14-3-3 We initially focused our study on MARK3, as co-operation with 14-3-3 to regulate substrates has mainly been described for this isoform. MARK3 is phosphorylated at T211 by LKB1 (Lizcano et al., 2004) and on T564 by aPKC (Hurov et al., 2004). In xPAR-1b (equivalent to hMARK2), aPKC phosphorylated T564 as well as a second C-terminal site (equivalent to S619 in hMARK3) (Kusakabe and Nishida, 2004). To investigate whether the LKB1 and aPKC phosphorylation sites are required for binding of 14-3-3 to hMARK3 (hereafter referred to as MARK3), Ala-mutants of these residues were expressed as glutathione S-transferase (GST)-fusion proteins in HEK 293 cells, and monitored for their ability to interact with endogenous 14-3-3 isoforms. As shown in Fig. 1, wild-type MARK3 bound to 14-3-3 and 14-3-3 , as determined by mass-fingerprinting (Fig. 1A), and this interaction was not disrupted by mutating the aPKC sites T564 and S619, alone or in combination (Fig. 1B). Mutation of T211, or of the catalytic residue D196, resulted in only a modest decrease in the ability of MARK3 to bind 14-3-3. In dPAR-1, a fragment encompassing the kinase- and UBAdomain reportedly bound 14-3-3 (Benton et al., 2002). However, an equivalently truncated version of MARK3 (termed kd+UBA) failed to bind 14-3-3 (Fig. 1B). As expected, mutation of T211 or D196 resulted in the loss of kinase activity, whereas mutation of T564 and/or S619 in MARK3 did not significantly affect T-loop phosphorylation (assessed using an antibody specific for phosphorylated T211) or kinase activity, monitored by employing the AMARA peptide substrate or the Cdc25c protein substrate. Binding of MARK3 to 14-3-3 requires phosphorylation and an intact phospho-Ser/Thr binding pocket of 14-3-3 Based on the study of three 14-3-3 point mutations and their ability to bind dPAR-1, Benton et al. suggested that the interaction of dPAR-1 wit (...truncated)