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.

To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulindependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P؉1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P؉1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P؉1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.Phosphorylation is a prevalent modification in cells that controls many functions such as signaling transduction, proliferation and apoptosis. It is estimated that at least one-third of all proteins in eukaryotic cell are phosphorylated at any given time (1, 2). More than 500 human protein kinases have been identified so far (3). A high degree of selectivity in substrate phosphorylation is necessary to maintain functional integrity of this very complicated signaling environment. Precision in phosphorylation is particularly critical when a substrate protein is phosphorylated at two (or more) phosphorylation sites, and those sites 1) are phosphorylated by distinct upstream kinases and 2) confer distinct properties on that substrate. To assure fidelity of signaling in this common situation, each upstream kinase must show high specificity by phosphorylating only the relevant phosphorylation site and not the inappropriate site(s). This requirement poses a major challenge in evolutionary design of kinase peptide specificity, since those upstream kinases are usually recruited to the substrate, and such recruitment can overcome much of the barrier provided by peptide specificity (4).These considerations raise the important issue as to what elements in kinase peptide specificity confer the strongest reciprocal specificity between kinases, i.e. which prevent one kinase from phosphorylating substrates phosphorylated by another. For Ser/Thr kinases, we propose that much of this reciprocal specificity is provided by the evolution of three broad classes (5): basophilic kinases that phosphorylate sites with clustered positive charges, acidophilic kinases that phosphorylate sites with clustered negative charges, and proline-directed kinases that ph...

Activation loop phosphorylation plays critical regulatory roles for many kinases. Unlike other protein kinase Cs (PKC), PKC-␦ does not require phosphorylation of its activation loop (Thr-507) for in vitro activity. We investigated the structural basis for this unusual capacity and its relevance to PKC-␦ function in intact cells. Mutational analysis demonstrated that activity without Thr-507 phosphorylation depends on 20 residues N-terminal to the kinase domain and a pair of phenylalanines (Phe-500/Phe-527) unique to PKC-␦ in/near the activation loop. Molecular modeling demonstrated that these elements stabilize the activation loop by forming a hydrophobic chain of interactions from the C-lobe to activation loop to N-terminal (helical) extension. In cells PKC-␦ mediates both apoptosis and transcription regulation. We found that the T507A mutant of the PKC-␦ kinase domain resembled the corresponding wild type in mediating apoptosis in transfected HEK293T cells. But the T507A mutant was completely defective in AP-1 and NF-B reporter assays. A novel assay in which the kinase domain of PKC-␦ and its substrate (a fusion protein of PKC substrate peptide with green fluorescent protein) were co-targeted to lipid rafts revealed a major substrate-selective defect of the T507A mutant in phosphorylating the substrate in cells. In vitro analysis showed strong product inhibition on the T507A mutant with particular substrates whose characteristics suggest it contributes to the substrate selective defect of the PKC-␦ T507A mutant in cells. Thus, activation loop phosphorylation of PKC-␦ may regulate its function in cells in a novel way. Protein kinase C (PKC)2 is a family of 9 genes that can be further divided into classical, novel, and atypical PKCs, depending on their structural characteristics and their requirement for activation (1, 2). Each of them is autoinhibited by an intramolecular interaction of the kinase domain with an N-terminal regulatory domain, whose organization differs between subfamilies. Classic PKCs have C1 and C2 domains that bind diacylglycerol and Ca 2ϩ , respectively, for their activation. Novel PKCs have a C1 domain that binds diacylglycerol, but their C2-like domain does not bind Ca 2ϩ . Atypical PKCs do not have the ability to bind either Ca 2ϩ or diacylglycerol, but are activated by other lipids or small G-proteins. Binding of the regulatory region with appropriate cofactors causes a conformational change that releases the autoinhibition and results in activation.Besides the co-factor-induced conformational change, PKC activity is also regulated by phosphorylation on its kinase domain, most importantly on its activation loop (3, 4). The activation loop is a stretch of 20 -30 amino acids located in the catalytic cleft of the kinase domain of all eukaryotic protein kinases that form part of the substrate peptide binding surface. The activation loop is relatively flexible, and undergoes varied forms of conformation regulation between the active and inactive states (5-7). One of the most common modes of k...

Three studies shed light on the decade-old observation that the actin cytoskeleton is hijacked to facilitate entry of HIV into its target cells. Polymerization of actin is required to assemble high concentrations of CD4 and CXCR4 at the plasma membrane, which promote viral binding and entry in both the simple model of infection by free virus and the more physiologically relevant route of infection through the virological synapse. Three types of actin-interacting proteins-filamin, ezrin/radixin/moesin (ERM), and cofilin-are now shown to play critical roles in this process. Filamin binds to both CD4 and CXCR4 in a manner promoted by signaling of the HIV gp120 glycoprotein. ERM proteins attach actin filaments to the membrane and may promote polymerization of actin. Early in the process of viral entry, cofilin is inactivated, which is proposed to facilitate the early assembly of actin filaments, but cofilin is reported to be activated soon thereafter to facilitate postentry events. This complex role of cofilin may help to reconcile the paradox that actin polymerization promotes initial binding and fusion steps but inhibits some subsequent early postentry events.

Background: Phosphatidylinositol 4,5-bisphosphate (PIP2) activates moesin via two binding sites whose roles are poorly understood. Results: Critical residues are identified in both sites and an inhibitory linker (FLAP) is characterized. Conclusion: Activation of moesin requires PIP2 binding to each site and release of the FLAP. Significance: The results fit a sequential activation model involving conformational change and interfacial release of FLAP.

Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4(+) and CD8(+) T lymphocytes expressed PAR-1 and that expression was increased in CD8(+) T cells from human immunodeficiency virus (HIV)-infected patients. Thrombin enhanced cytokine secretion in CD8(+) T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8(+) T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines.