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...

ERM (ezrin, radixin moesin) proteins in lymphocytes link cortical actin to plasma membrane, which is regulated in part by ERM protein phosphorylation. To assess whether phosphorylation of ERM proteins regulates lymphocyte migration and membrane tension, we generated transgenic mice whose T-lymphocytes express low levels of ezrin phosphomimetic protein (T567E). In these mice, T-cell number in lymph nodes was reduced by 27%. Lymphocyte migration rate in vitro and in vivo in lymph nodes decreased by 18% to 47%. Lymphocyte membrane tension increased by 71%. Investigations of other possible underlying mechanisms revealed impaired chemokine-induced shape change/lamellipod extension and increased integrin-mediated adhesion. Notably, lymphocyte homing to lymph nodes was decreased by 30%. Unlike most described homing defects, there was not impaired rolling or sticking to lymph node vascular endothelium but rather decreased migration across that endothelium. Moreover, decreased numbers of transgenic T cells in efferent lymph suggested defective egress. These studies confirm the critical role of ERM dephosphorylation in regulating lymphocyte migration and transmigration. Of particular note, they identify phospho-ERM as the first described regulator of lymphocyte membrane tension, whose increase probably contributes to the multiple defects observed in the ezrin T567E transgenic mice. (Blood. 2012;119(2):445-453) IntroductionNormal immune function depends on lymphocytes in circulation binding to vascular endothelium, transmigrating across the endothelium, and migrating within tissue. 1-3 Lymphocyte migration and transmigration depend on cytoskeletal reorganization, including especially the actin cytoskeleton. However, linkage between plasma membrane and actin cytoskeleton is a potentially important aspect, which has not yet been well studied. Ezrin-radixin-moesin (ERM) proteins are a trio of very closely related human paralogs whose primary function is mediating linkage between the plasma membrane and cortical actin, which is the shell of polymerized actin that lies just below the membrane. 4,5 One of the most fundamental aspects of ERM protein function is their ability to regulate that linkage by switching between active and inactive conformations. In the active conformation, the N-terminal region, the FERM domain, binds to plasma membrane lipids and cytoplasmic tails of transmembrane proteins and the C-terminal region binds to F-actin. However, in the dormant conformation, those 2 regions bind intramolecularly to each other and therefore cannot mediate linkage via intermolecular interactions. The conformational switch between dormant and active forms is initiated and sustained by ERM protein binding to PI(4,5)P2 in the plasma membrane. [4][5][6][7] In addition, C-terminal phosphorylation plays an important role in stabilizing the active conformation. Solved structures of the dormant ERM protein elucidate the mechanism whereby phosphorylation stabilizes the active conformation. The critical threonine that is phosphorylated ...

In contrast to ATM-null mice, mice expressing a kinase-dead ATM variant exhibit embryonic lethality, associated with greater deficiency in homologous recombination.