LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation

Abstract

ERM (ezrin-radixin-moesin) proteins mediate linkage of actin cytoskeleton to plasma membrane in many cells. ERM activity is regulated in part by phosphorylation at a C-terminal threonine, but the identity of ERM kinases is unknown in lymphocytes and incompletely defined in other mammalian cells. Our studies show that lymphocyte-oriented kinase (LOK) is an ERM kinase in vitro and in vivo. Mass spectrometric analysis indicates LOK is abundant at the lymphocyte plasma membrane and immunofluorescence studies show LOK enrichment at the plasma membrane near ERM. In vitro peptide specificity analyses characterize LOK as a basophilic kinase whose optimal substrate sequence resembles the ERM site, including unusual preference for tyrosine at P-2. LOK's activity on moesin peptide and protein was comparable to reported ERM kinases ROCK and PKC but unlike them LOK displayed preferential specificity for moesin compared to traditional basophilic kinase substrates. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have >50% reduction in ERM phosphorylation. The findings on localization and specificity argue that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations fit the current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of importance in lymphocytes and confirm the role of ERM phosphorylation in regulating cell shape and motility.ezrin ͉ kinase specificity ͉ knockout ͉ migration ͉ moesin T he ERM family in mammals consists of 3 closely related members: ezrin, radixin and moesin whose major function is to link cortical actin filaments to the plasma membrane (1-4). ERM N terminus (the FERM/band 4.1 domain) binds to plasma membrane both by direct interaction with phospholipids and by binding cytoplasmic tails of transmembrane proteins such as CD43, CD44, and ICAMs. ERM C terminus (''tail'') binds to filamentous actin. ERMs exist not only in this active conformation, but also in an inactive conformation where the C terminus binds to the FERM domain, thereby blocking binding sites on both FERM and tail. There is an evolutionarily conserved phosphorylation site near the C terminus whose phosphorylation contributes to stabilizing the active conformation. In mitotic cells ERM phosphorylation is critical for achieving spherical morphology and rigidity (5, 6). For lymphocytes circulating in blood, ERM phosphorylation is understood to contribute to rigidity and maintenance of microvilli. In response to chemotactic factors (especially chemokines) those lymphocytes transition into flexible migrating cells concurrent with rapid extensive dephosphorylation of ERM, which facilitates their polarization (7-10).Given the importance of ERM phosphorylation, it is essential to...

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis

“…However, this fragment is devoid of kinase activity because it cannot phosphorylate ERM in transfected cells or either histone H2A or the moesin C-terminal domain in in vitro kinase assays. In contrast, we have shown previously and confirmed in this study that the longer fragment extending from the N terminus to amino acids 422 (LOK 422) is an active kinase (5). This suggests that the sequence between amino acids 332 and 422 is a regulatory domain.…”

“…Furthermore, in vitro peptide specificity analyses have identified an optimal LOK substrate sequence similar to the ezrin, radixin, and moesin (ERM) phosphorylation sites. Genetic evidence confirms that ERM are LOK substrates in lymphocytes because LOK knockout mice display strongly reduced ERM phosphorylation at a C-terminal site (5). The major function of ERM is to create links between the plasma membrane and cortical actin filaments.…”

“…In response to chemokines, dynamic dephosphorylation of ERM participates in a transition to a flexible, migrating cell form (10). Increased migration efficiency in LOK-deficient lymphocytes is consistent with rearrangements of the actin cytoskeleton resulting from ERM dephosphorylation (5). LOK deficiency also leads to increased LFA-1-mediated lymphocyte adhesion (11).…”

Model membranes to shed light on the biochemical and physical properties of ezrin/radixin/moesin

“…On one hand, several physiological processes involve ERM phosphorylation on conserved threonine residue in the actin binding site (T567 in ezrin, T564 in radixin, T558 in moesin). For example, in cellulo, moesin is phosphorylated by Rho kinase during platelet activation [53,54] or by lymphocyte-oriented kinase (LOK) which stimulates lymphocyte migration and polarization [55]. A number of other kinases in vertebrate cells can phosphorylate ERMs on this regulatory threonine, including protein kinase C (PKC) α and θ, Nck-interacting kinase (NIK), human serine/threonine kinase (Mst4) [56][57][58][59].…”

Ezrin is a negative regulator of death receptor-induced apoptosis

“…ERM are activated by phosphorylation at the C-terminal threonine or by the binding of phosphatidylinositol 4,5-biphosphate (PIP2) (Bretscher et al, 2002;Niggli and Rossy, 2008;Belkina et al, 2009). Activated ERM proteins bind directly to the cortical actin cytoskeleton.…”