Personal pdf file for E. Peverelli, E. Giardino, E. Vitali, D. Treppiedi, A. G. Lania, G. Mantovani

Transcription

1 Personal pdf file for E. Peverelli, E. Giardino, E. Vitali, D. Treppiedi, A. G. Lania, G. Mantovani With compliments of Georg Thieme Verlag Filamin A in Somatostatin and Dopamine Receptor Regulation in Pituitary and the Role of camp/pka Dependent Phosphorylation DOI /s Horm Metab Res 2014; 46: For personal use only. No commercial use, no depositing in repositories. Publisher and Copyright 2014 by Georg Thieme Verlag KG Rüdigerstraße Stuttgart ISSN Reprint with the permission by the publisher only

2 Review 845 Filamin A in Somatostatin and Dopamine Receptor Regulation in Pituitary and the Role of camp/pka Dependent Phosphorylation Authors E. Peverelli 1, E. Giardino 1, E. Vitali 2, D. Treppiedi 1, A. G. Lania 3, G. Mantovani 1 Affiliations 1 Endocrine Unit, Department of Clinical Sciences and Community Health, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy 2 Laboratory of Cellular and Molecular Endocrinology, Humanitas Research Center, Rozzano, Italy 3 Endocrine Unit, IRCCS Humanitas Clinical Institute, Rozzano, University of Milan, Milan, Italy Key words cytoskeleton pituitary tumors GPCR received accepted Bibliography DOI /s Published online: July 28, 2014 Horm Metab Res 2014; 46: Georg Thieme Verlag KG Stuttgart New York ISSN Correspondence M. Giovanna, MD, PhD Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico Unità Operativa di Endocrinologia e Diabetologia University of Milan Padiglione Granelli via F. Sforza Milano Italy Tel.: + 39/02/ Fax: + 39/02/ giovanna.mantovani@unimi.it Abstract Molecular mechanisms underlying resistance of pituitary tumors to somatostatin (SS) and dopamine (DA) analogues treatment are not completely understood. Resistance has been associated with defective expression of functional somatostatin and dopamine receptors SSTR2, SSTR5, and DRD2, respectively. Recently, a role of cytoskeleton protein filamin A (FLNA) in DRD2 and SSTR receptors expression and signaling in PRL- and GH-secreting tumors, respectively, has been demonstrated, first revealing a link between FLNA expression and responsiveness of pituitary tumors to pharmacological therapy. No molecular events underlying the reduction of FLNA levels in resistant tumors have been so far identified. Introduction Pituitary tumors are rare neoplasia that derive from excessive proliferation of each subtype of pituitary cells and present with specific endocrine syndromes or mass symptoms or both. The main G protein coupled receptors (GPCRs) target of pharmacological treatment of pituitary tumors are the dopamine (DA) receptor DRD2 and somatostatin (SS) receptors SSTR2 and 5. In particular, DRD2 agonists are used in the treatment of prolactin (PRL)- and, to a lesser extent, ACTH-secreting and nonfunctioning pituitary tumors (NFPA), whereas long acting SS analogues, such as octreotide and lanreotide, are currently used in the treatment of pituitary tumors, particularly GHand TSH-secreting tumors [1, 2]. Prolactinomas are the most frequent pituitary tumors and DA agonists normalize PRL levels and reduce tumor size in the majority of patients by binding DRD2, the DA receptor subtype expressed in pituitary lactotrophs (reviewed in [1]). DRD2 FLNA can be phosphorylated by PKA on Ser2152, with increased FLNA resistance to cleavage by calpain and conformational changes affecting FLNA regions involved in SSTR2 and DRD2 binding and signal transduction. In this respect, the effect of camp/pka pathway in the regulation of FLNA stability and/or function by modulating its phosphorylation status could assume particular importance in pituitary, where camp cascade plays a crucial role in pituitary cell functions and tumorigenesis. This review will discuss the role of FLNA in the regulation of the main GPCRs target of pharmacological treatment of pituitary tumors, that is, SSTR2 and DRD2, focusing on the effects of camp/pka-mediated FLNA phosphorylation on FLNA biological functions. couples to Gi/Go proteins to inhibit adenylyl cyclase activity and reduce intracellular camp levels. Somatostatin analogues are currently used to successfully treat acromegalic patients since they inhibit cell proliferation and hormone secretion by binding with high affinity SSTR2 and, to a lesser extent, SSTR5, which are both expressed at high density in most GH-secreting pituitary tumors [3, 4]. By coupling with multiple PTXsensitive G proteins, SSTs inhibit adenylyl cyclase activity and some subtypes reduce calcium entry by modulating L-type Ca 2 + and K + channels, all these events being involved in the reduction of hormone secretion. Both SSTR2 and SSTR5 mediate the antiproliferative effects of SS, by tyrosine phosphatase activation and ERK1/2 phosphorylation inhibition, respectively, whereas only SSTR2 and SST3 subtypes mediate apoptotic effects [5 8].

3 846 Review Mechanisms of Resistance of Pituitary Adenomas to Pharmacological Treatment To date, the promises raised by the large bulk of preclinical data concerning the effects of long acting SS analogues and DRD2 agonists on hormone secretion and cell proliferation have been only in part fulfilled. Indeed, clinical practice demonstrated a great variability in the frequency and entity of favorable responses during chronic treatment of patients with pituitary tumors with these agents. In particular, 10 % of patients with prolactinoma and 30 % of patients with acromegaly are resistant to cabergoline and octreotide, respectively, while most NFPA and ACTH secreting adenomas are unresponsive to both drugs [9 12], with a major impact on mortality, morbidity, and quality of life. Moreover, the occurrence of resistance to DRD2 agonists has been associated with the shift of a prolactinoma to an invasive tumor or even a carcinoma [12]. Despite intensive research, the molecular events involved in pituitary tumors resistance to pharmacological treatment are still unclear. Concerning resistance to dopaminergic drugs, reduction in DRD2 expression has been reported in PRL-secreting tumors, particularly in DA-resistant with respect to DA-sensitive tumors [13 15], but the molecular determinants of this low expression have not been identified. No mutation of the DRD2 gene has been found in prolactinomas [16], the only finding being a correlation of some DRD2 polymorphisms with DA resistance [17]. More controversial is the association of SS resistance to the reduction of SSTR expression, since some tumors are resistant to therapy despite high SSTR2 expression [18 25]. Also in this case, the molecular events responsible for reduced SST expression are still largely unknown, mutations in their coding sequence and/ or loss of heterozygosity in loci where they are located being very rare events [26 29]. The hypothesis of post-receptor alterations involved in resistance to SS analogues is supported by the dissociation between the antisecretory and antiproliferative effects of SS analogues observed in some acromegalic patients [30]. Alterations in SSTRs signal transduction, involving AIP and ZAC1, have been investigated. Interestingly, acromegalic patients Scaffold region Rep. 21 (integrin interaction) Rep (SSTR2 interaction) Rep. 19 (DRD2 interaction) Rep. 1 ABD Hinge1 Rep. 24 (Dimerization) Hinge2 Ser2152 Rod 2 FLNA monomer Rod 1 Fig. 1 Schematic representation of a FLNA dimer. The actin-binding domain (ABD) at the N-terminus is shown. The rod-1 domain (repeats 1 15), rod-2 domain (repeats 16 23), and repeat 24 are separated by 2 hinge regions. Regions involved in the interaction with partner proteins are shown. Black circles: repeats involved in DRD2/SST2 binding. Gray circles: scaffold region. The repeat 24 mediates FLNA homodimerization. The serine residue target of PKA-mediated phosphorylation is indicated. with AIP germline mutations or tumors with low levels of AIP are typically resistant to SS analogues [31]. Growing evidence revealed that GPCR expression, localization, and signaling are regulated by interaction with different cytoskeleton proteins, including filamin A (FLNA) [32], suggesting another possible mechanism underlying drug resistance. This actin-binding protein is involved in the regulation of expression and signaling of DRD2 and SSTR2, with important consequences for pituitary tumor responsiveness to drugs targeting these receptors. FLNA Role in Pituitary Tumor Responsiveness to DRD2 Agonists and Somatostatin Analogues FLNA structure and functions Although originally it was believed that GPCRs mediate signal transduction only through the activation of coupled G proteins, it is now well established that these receptors directly interact through their intracellular loops with cytoplasmic and surface proteins involved in GPCRs stabilization, desensitization, internalization, and signal transduction. Among these, arrestins are well known as both GPCR signal terminators and signal transducers, and more recently also cytoskeleton proteins have been recognized to play a considerable role. Indeed, cytoskeleton not only plays a paramount role in cell morphology maintenance, cell migration and adhesion, cell division and organelles localization and movement within the cytosol but it also participates in extracellular signal transduction and regulates activity of several receptors. The 3 main structural components of the cytoskeleton are microtubules, intermediate filaments and microfilaments, resulting from the polymerization of different monomers, which undergo continual turnover and rearrangement and are specifically associated with a number of partner proteins that determine the structural and functional differences. In particular, microfilaments consist of actin filaments that are polymerized, depolymerized, and cross-linked into bundles and networks with the help of multiple families of cytoskeletal specific actin binding proteins. Filamins belong to the family of the actin cross-linking proteins, characterized by a conserved actin binding domain followed by a rod-like domain that allows to dimerize. Some of these proteins such as α-actinin form parallel actin bundles, whereas filamins form orthogonal networks. The filamin family consists of 3 homologous high-molecular weight proteins, FLNA, FLNB, and FLNC, encoded by genes located on different chromosomes (chr.x, chr.3, and chr.7, respectively). The 3 isoforms of filamin in mammals show a strong homology in the entire sequence and possess highly conserved genomic organization [33]. Human FLNA, mapping to Xq28, is the first actin filament cross-linking protein identified in nonmuscle cells [34] and is the most abundant filamin isoform in adults. The expression of FLNC is restricted to skeletal and cardiac muscle, whereas FLNA and B are ubiquitously expressed. Several studies suggest that filamins are essential for normal human development, and mutations in the respective genes cause a wide range of developmental malformations of the brain, bone, and heart with moderate to lethal consequences [35]. In particular, FLNA mutations cause periventricular nodular heterotropia, a brain malformation due to abnormal neuronal migration, in which a subset of neurons fails to migrate into the developing cerebral cortex [36] or a wide spectrum of con-

4 Review 847 genital malformations, such as otopalotodigital syndrome, frontometaphyseal dysplasia and Melnick Needles syndrome [37]. FLNA is composed of 2 subunits of 280 kda each that self-assemble. A schematic representation of a FLNA dimer structure is shown in Fig. 1. Each monomer possesses an actin-binding domain (ABD) at the N-terminus, which consists of 2 calponin homology domains, followed by 24 immunoglobulin (Ig)-like repeats of about 96 amino acid residues. Two calpain-sensitive hinge regions separate the 24 repeats in a rod-1 domain (repeats 1 15), rod-2 domain (repeats 16 23), and repeat 24. A secondary ABD of lower affinity is located in the rod-1 domain, whereas rod-2 is involved in the interaction with partner proteins. The repeat 24 is the self-association domain that mediates FLNA homodimerization, allowing the formation of V-shaped flexible structures, which results in the perpendicular cross-linking of actin filaments. Besides stabilizing actin filaments in a three-dimensional structure, FLNA anchors several transmembrane proteins, such as integrins, ion channels, and several GPCRs with which it directly interacts, to the actin cytoskeleton [32]. In addition, FLNA functions as important signaling scaffold by binding a variety of proteins, including receptors, ion channels, intracellular signaling molecules, kinases, and transcription factors (reviewed in [38]). These interactions are regulated by phosphorylation events, proteolysis, mechanical forces, competition, and multimerization of partners. Role of FLNA in the regulation of DRD2: relevance for PRL-secreting pituitary tumors Li and colleagues first demonstrated FLNA interaction with the third cytoplasmic loop of DRD2 [39]. The specificity of this interaction was identified in a yeast 2-hybrid screen and confirmed by protein binding. They also showed that this association enhances coupling efficiency of DRD2 to adenylate cyclase and plays a role in cell surface receptor clustering by using as cell model 2 human melanoma cell lines, M 2, that does not express FLNA and A 7, stably transfected with FLNA. Almost simultaneously, another study using the third intracellular loop of the DRD2 as bait in a yeast 2-hybrid approach to screen a human brain cdna library, confirmed a specific DRD2/FLNA association, better defining the specific regions of FLNA (repeat 19) and DRD2 (amino acids in the N-terminal region of the third intracellular loop) involved [40]. FLNA and DRD2 co-localized in cell cultures of rat striatum. The authors found that DRD2 was predominantly intracellular in M 2 cells, whereas it localized at the plasma membrane in A 7 cells, suggesting that FLNA is required for the cell surface localization of DRD2. Further confirmation to this hypothesis came from the observation that the overexpression of a dominant negative truncated form of FLNA (repeats 18 19, containing the DRD2, but not the actin, binding domain) caused a marked reduction in both the number and half-life of cell surface DRD2 receptors [41]. The finding that human prolactinomas, and in particular those removed from patients in whom DA analogues treatment did not achieve PRL normalization and tumor shrinkage, showed low levels of both FLNA and DRD2 [42], is the first observation suggesting a role for FLNA in DRD2 regulation in this type of tumor. Alterations of FLNA levels in primary cultured prolactinoma cells by gene silencing or overexpression resulted in corresponding modifications of DRD2 levels, demonstrating that these 2 events are causally related [42] ( Fig. 2a). Moreover, DRD2 signal transduction, including reduction of PRL release and ERK1/2 phosphorylation, was impaired after FLNA silencing, whereas DA-resistant prolactinomas lacking FLNA recovered PRL responsiveness when transfected with FLNA expression vector [42]. The authors further investigated the mechanisms by which FLNA regulates DRD2 in a cell model of prolactinoma endogenously expressing functional DRD2 and FLNA, that is, MMQ cells. Data show that FLNA is not only required for DRD2 targeting to the cell membrane, but also protects DRD2 against lysosomal degradation, suggesting a role for FLNA in the control of DRD2 fate towards recycling processes or lysosomal degradation [42] ( Fig. 2b, c). Since FLNA directly interacts with beta arrestins [43] that are involved in DRD2 trafficking [44], it is possible to hypothesize the formation of a complex receptor-flnaarrestin involved in the regulation of DRD2 stability. This protective effect of FLNA from instability has been demonstrated for other receptors, such as calcium-sensing receptor, calcitonin receptor, cystic fibrosis transmembrane conductance regulator, and the high-affinity IgG receptor FcgammaRI [45 48]. These data strongly demonstrate a structural role of FLNA in anchoring DRD2 to actin cytoskeleton and regulating receptor localization and stability, but also suggest an additional functional role of FLNA as scaffold for signaling molecules involved in DRD2 signal transduction. Indeed, since the pituitary has a substantial DRD2 reserve for PRL inhibition, and PRL response reaches the plateaux at about 40 % receptor occupancy in rat pituitary cells [49], the 60 % reduction of DRD2 levels measured in prolactinomas and MMQ cells after FLNA knockdown might not entirely account for the loss of D2R effects on PRL release and cell proliferation. Overall, FLNA is crucial for DRD2 expression and signaling in lactotrophs and the loss of FLNA expression may be one of the mechanisms involved in loss of DA responsiveness in human prolactinomas. Up to now, the molecular events underlying FLNA reduced expression are unknown, the only observation being the absence of alterations in the CpG island with the highest probability to have regulatory functions, excluding epigenetic silencing [42]. Role of FLNA in the regulation of SSTR2: relevance for GH-secreting pituitary tumors Recently, it has been shown by surface plasmon resonance (SPR) that FLNA directly interacts with SSTR2 first intracellular loop [50]. Searching for the FLNA region responsible of this binding, the authors found that only FLNA repeats bound SSTR2 in a dose-dependent manner and with a high affinity. By co-immunoprecipitation experiments, they also showed that FLNA/SSTR2 interaction occurs in cellulo in different cell lines, such as neuroendocrine pancreatic BON, keratinocyte HaCaT cells, or CHO transfected with SSTR2. FLNA is required for SSTR2-mediated inhibition of cell survival, and the molecular mechanism involves a competition of FLNA with p85, the regulatory subunit of PI3K, for direct binding to SSTR2. Ligand-stimulated FLNA binding results in the disruption of the SSTR2-p85 complex and the subsequent inhibition of PI3K [50]. The authors also found enhanced ligand-induced SSTR2 internalization rate in FLNA-deficient M 2 cells with respect to A 7 cells, suggesting FLNA requirement for SSTR2 stabilization at the cell membrane. Interestingly, SSTR2 was correctly targeted to the plasma membrane in M 2 cells in the absence of agonist, contrary to DRD2, that remained mainly cytoplasmatic in M 2 cells [40], implying different mechanisms involved.

5 848 Review Fig. 2 DRD2 expression and localization in prolactinomas depends on FLNA levels. a Representative immunoblots of FLNA and DRD2 in DA-sensitive or resistant prolactinoma cells transiently transfected (72 h) with negative control or FLNA sirna or prep4-flna. FLNA silenced cells showed a strong decrease in FLNA protein expression that was associated with a reduction of DRD2 expression, whereas FLNA overexpression in DA-resistant tumors was associated with a significant increase of DRD2 expression. The equal amount of protein was confirmed by stripping and reprobing with an anti-gapdh antibody. b Representative immunoblot of DRD2 in MMQ cells transiently transfected with negative control or FLNA sirna. FLNA sirna treated cells showed a strong reduction of DRD2. The lysosome inhibitor chloroquine induced an increase in DRD2 expression in FLNA silenced cells with respect to control cells. The equal amount of protein was confirmed by stripping and reprobing with an anti-gapdh antibody. c Left panel. Representative confocal microscopy images of MMQ cells transfected with negative control sirna or FLNA sirna cells for 72 h stained for DRD2. In control cells DRD2 was mainly localized at the plasma membrane, with frequent clustering, whereas in FLNA sirna cells, DRD2 was redistributed to cytoplasmic vescicles. Right panel. Biochemical analysis of membrane expression of DRD2. Seventy-two hours after sirna transfection, biotinylated cell surface proteins and total cellular proteins were immunoprecipitated by DRD2 antibody and cell surface DRD2 detected by an antibiotin antibody. Biotinylation assay showed reduced DRD2 expression at the cell membrane in cells transfected with FLNA sirna for 72 h. The graph shows the quantification of cell surface expression of DRD2. *p < 0.05 vs. negative control, t-test [42]. (Color figure available online only). Accordingly, recently published data from our lab showed that FLNA expression in human GH-secreting tumors did not correlate with SSTR2 levels, and FLNA silencing in human somatotroph tumoral cells did not affect SSTR2 expression and membrane localization [51]. However, overexpression of a FLNA dominant negative truncated mutant that specifically prevents SSTR2-FLNA binding reduced SSTR2 expression after prolonged agonist exposure in rat GHsecreting cell line GH3, suggesting that FLNA is involved in SSTR2 stabilization. Moreover, our silencing experiments showing that FLNA is required for SSTR2-induced reduction of cyclin D1 and caspase3/7 activation in tumoral somatotrophs [51] suggest a crucial role for FLNA in mediating antiproliferative and proapoptotic signaling of SSTR2, consistent with the view that FLNA participates to signal transduction as scaffold protein for signaling molecules. All these data support a new role for FLNA in the responsiveness of patients with GH-secreting pituitary tumors to pharmacological treatment with SS analogues. Low levels of FLNA, causing loss of coupling of SSTR2 with downstream signal transduction molecules, might explain the resistance to SS analogues in SSTR2 expressing tumors. camp/pka Pathway Activation: Effects on FLNA Regulation Role of camp in pituitary functions camp is a universal second messenger that regulates numerous cell functions, including cell proliferation, in response to GPCRs activation [52]. It is well known that camp signaling cascade plays a central role in pituitary cell functions, and camp/pka pathway alterations are involved in pituitary tumorigenesis (reviewed in [53]). In particular, the promitogenic effects of camp on pituitary somatotrophs have been broadly supported by several studies, in agreement with the notion that mutations activating camp formation or signaling are associated with the occurrence of sporadic or familial acromegaly. Indeed, somatic activating mutations in GNAS1 gene, encoding the α subunit of the stimulatory G protein (Gsα) (gsp oncogene) [54, 55] are found in % of GH-secreting pituitary tumors. However, gsp mutations show a reduced oncogenic potential and associate with a benign phenotype due to different intracellular regulatory mechanisms able to counteract in vivo the activation of the camp pathway, such as the increased expression of phosphodiesterases that degrade camp [56, 57].

6 Review 849 In contrast to the mitogenic effect of camp in the pituitary somatotrophs, camp exerts inhibitory effects on the proliferation of gonadotroph and lactotroph derived cells [58, 59], in line with the notion that mutations activating camp pathway are not associated with the occurrence of NFPAs, that are mainly constituted by gonadotroph derived cells, and of PRL-secreting pituitary tumors. These divergent effects of camp in pituitary cells of different lineages are mediated by the 2 main camp effectors, PKA and Epac [59], and are associated with its ability to positively or negatively regulate the mitogen-activated protein kinase (MAPK) signaling pathways. The most studied camp downstream effector is PKA. This serine/ threonine kinase is a tetrameric enzyme composed of 2 regulatory (R) and 2 catalytic (C) subunits. The R subunit exists in 2 isoforms, R I and R II, which give rise to 2 PKA isozymes (PKA I and II). After binding of camp to each R subunit, C subunits dissociate from the R subunits and phosphorylates in the nucleus the transcription factor CREB at Ser 133. The active CREB binds to the conserved consensus camp response element (CRE) as a dimer, leading to transactivation of responsive genes [60]. Interestingly, an increased Ser 133 phosphorylation of CREB has been found in human somatotroph tumors compared to NFPAs [61], suggesting an increased transcription of specific CREB-dependent target genes in this type of tumors. Finally, camp pathway, interacting with calcium signaling, plays a major role in the regulation of hormone synthesis and secretion in pituitary in response to hypothalamic neuroendocrine regulatory factors. PKA-mediated FLNA phosphorylation FLNA binding to its multiple interacting partners is regulated by post-translational modifications. FLNA is known to be a phosphoprotein, whose phosphate content ranges from 18 to 40 mol of Pi/mol FLNA [62], but the biological significance of phosphorylation is still unclear. Many kinases, such as PKA, PKC, CaMkinase II, Pak1 (p21-activated kinase 1), RSK (ribosomal S6 kinase), and cyclin B1/Cdk1 [63 67] can phosphorylate FLNA. Chen et al. demonstrated that FLNA is phosphorylated by PKA in platelets, with a consequent stabilization of FLNA against proteolysis by calpain [63]. In particular, the N-terminal region of FLNA contains a PKA site probably involved in F-actin interaction [68], whereas the only PKA phosphorylation site in the C-terminal region of FLNA, containing the calpain cleavage site, is serine 2152 in the repeat 20 of FLNA [65]. The in vivo phosphorylation of this residue has been demonstrated after PKA activation [69 71]. Serine 2152, located in a serine-proline motif, is also a target of Pak1 and RSK, whereas the complex cyclin B1/Cdk1 phosphorylates serine 1436 [67]. The biological effects of FLNA phosphorylation are not fully elucidated, most of the studies focusing on regulation of FLNA proteolysis and cell migration. Phosphorylation effects on FLNA proteolysis Calpain family includes intracellular calcium-dependent structurally related cysteine proteases that are widely expressed in various tissues, and play important roles in the regulation of a variety of cellular functions, such as intracellular signaling, proliferation, differentiation, and apoptosis. Calpain-mediated proteolysis is involved in hormone maturation and secretion as well as cell signaling in the pituitary [72 74]. The calpain cleavage site of FLNA has been localized in the 2 hinge regions, one at residues , 100 KDa away from the C-terminus, and another at 10 KDa from C-terminal domain, producing fragments of different molecular weight (180, 100, 90, and 10 KDa) ( Fig. 3) [33]. The biological effects of FLNA proteolysis by calpain are multiple and still under investigation. In melanoma FLNA-expressing A 7 cells, Western blot analysis detected FLNA proteolitic fragments of the molecular weight expected from calpain cleavage that are reduced after incubation with calpain inhibitor calpeptin, but Fig. 3 FLNA proteolysis by calpain is regulated by Ser 2152 phosphorylation. Schematic representation of a FLNA monomer. Ser2152 residue location and target sites for calpain cleavage in the 2 hinge regions are shown. The FLNA proteolitic fragments of different molecular weight that derive from calpain-mediated proteolysis (180, 100, 90, and 10 KDa, respectively) are schematically shown.

7 850 Review not with caspase-3/7 inhibitor, Ac-DEVD-CHO [75]. The authors showed that calpain-mediated FLNA cleavage was significantly enhanced by hypoxia and that the C-terminal proteolitic fragment enhances nuclear accumulation and the transactivation function of hypoxia-inducible factor-1α. The 90 kda FLNA fragment interacted with the androgen receptor and translocated to the nucleus where it inhibited AR transcriptional activity [76, 77] and promoted sensitization of prostate cancer cells to anti-androgen therapy [78]. Moreover, calpain cleavage is involved in the regulation of cell migration processes, and the control of focal adhesion turnover by FLNA is calpain-dependent [79]. Thus, understanding the events, such as phosphorylation that regulate FLNA cleavage by calpain are of great relevance to gain further insights into the molecular mechanisms in which FLNA and its proteolitic fragments are involved. It has been demonstrated that camp pathway activation confers an increased resistance of FLNA to calpain cleavage, but does not protect against other proteases such as trypsin, papain or thermolysin [63, 64]. FLNA recovered its sensitivity to calpain after FLNA incubation with protein phosphatases purified from platelets [63]. These data suggest that PKA-mediated Serine 2152 phosphorylation induces a conformational change of FLNA, resulting in a structure that prevents calpain attack. A critical role in the FLNA phosphorylation/dephosphorlyation processes, regulating FLNA susceptibility to proteolysis by calpain, is played by calcium/calmodulin-dependent threonine/serine protein phosphatase calcineurin. FLNA Serine 2152 is a substrate for calcineurin in vivo and in vitro, and the calcineurin specific inhibitor cyclosporine A protects FLNA in platelets against calpain cleavage, by increasing phosphorylation status of FLNA [80]. FLNA phosphorylation and effects on cell migration FLNA role in cell migration remains controversial, depending on FLNA expression levels and interacting partners. A direct involvement of FLNA Serine 2152 phosphorylation in cell migration has been recently demonstrated. Forskolin stimulation of CHP100 neural cells induced FLNA serine 2152 phosphorylation and a reduction in cell migration [71], these effects being reproduced by the overexpression of a phosphomimetic S2152D FLNA mutant. FLNA serine 2152 phosphorylation is also induced by the loss of ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2), another gene whose mutations are associated with periventricular heterotopias. The authors also showed that phosphorylated FLNA redistributed from the plasma membrane to the cytoplasm, with alterations in focal adhesion organization and distribution, suggesting a mechanism involved in the control of neuronal migration in periventricular heterotopias. Another effect of FLNA phosphorylation is the regulation of integrin binding by FLNA. FLNA has been shown to interact through the repeat 21 with the beta subunit cytoplasmic domain of several integrins (β1, β2, β3, β7) [81 85] with consequent relevant effects on cell migration. In particular, a strong FLNA-integrins interaction inhibits cell migration by a mechanism involving membrane protrusion and cell polarization inhibition [81]. Crystallographic studies have shown that FLNA repeat 20 is partially unfolded and brings repeat 21 in close proximity to repeat 19 [86]. This autoinhibitory structure sterically prevents the binding of FLNA repeats 21 to integrins. Recently it has been demonstrated by molecular dynamic simulations that tensile force coupled to FLNA phosphorylation at serine 2152 induces a complete dissociation of autoinhibition [87]. These data indicated that FLNA phosphorylation facilitates the integrin binding by decreasing the mechanical force required for dissociation of FLNA autoinhibitory structure, although the phosphorylation itself without tension is not sufficient to remove the autoinhibition. camp Pathway and FLNA Phosphorylation: A Possible Involvement in Drug Resistance of Pituitary Tumors? Based on these data, it is possible to hypothesize that the regulation of FLNA phosphorylation status is a mechanism involved in pituitary responsiveness to pharmacological therapy. Although camp pathway plays a crucial role in pituitary cell functions and although FLNA is involved in pituitary tumors sensitivity to drugs by regulating DRD2 and SSTR2, no data are still available about a possible involvement of camp/pka in the regulation of FLNA stability and/or function by modulating its phosphorylation status in pituitary. First, based on the observation that FLNA Serine 2152 phosphorylation protects FLNA from calpain-dependent degradation, it Fig. 4 Schematic representation of the role played by FLNA in DRD2 and SSTR2 expression and signaling. Each monomer of a FLNA dimer directly interacts through repeat 20 with DRD2 third intracellular loop and SSTR2 first intracellular loop. This interaction is required for DRD2 plasma membrane localization and stability, and also for DRD2 signal transduction. On the contrary, FLNA binding is not required for SSTR2 targeting to the plasma membrane, but it is crucial for SSTR2 stabilization at the cell membrane after agonist stimulation and for SSTR2 signal transduction. The coupling of both receptors to inhibitory G proteins (Gαi) leading to adenylyl cyclase inhibition is shown. The possible effects of Ser 2152 phosphorylation in the repeat 20 of FLNA by PKA on FLNA functions are shown in detail.

8 Review 851 is conceivable to speculate that camp pathway activation and increased PKA activity might lead to increased levels of fulllength FLNA and decreased production of proteolitic FLNA fragments, whose biological activities in pituitary are unknown. Since FLNA is required for expression and signaling of SSTR2 [50, 51] that mediates inhibitory effects on cell proliferation and hormone secretion, a FLNA increase after camp pathway activation might be one of the intracellular regulatory mechanisms able to counteract in vivo the activation of the camp pathway in gsp positive GH-secreting pituitary tumors and responsible for the reduced oncogenic potential of these mutations. Moreover, phosphorylation-dependent FLNA stabilization might explain the increased responsitivity of gsp positive tumors to treatment with SS analogues, a feature up to now unexplained, since no increase of SSTRs has been found in these tumors [26, 88, 89]. Moreover, it is possible to hypothesize that the phosphorylation status of FLNA regulates also the ability to bind DRD2 and SSTR2 ( Fig. 4). Indeed, since FLNA serine 2152 phosphorylation decreases the force required for dissociation of autoinhibitory structure involving repeats , it might also facilitate the binding of FLNA repeats to SSTR2 or DRD2, as demonstrated for integrin binding [87]. Similarly, since the repeat 21 is located in the scaffold region of FLNA, crucial for the efficiency of signal transduction, Ser2152 phosphorylation may regulate FLNA scaffold function by altering the ability of FLNA to bind proteins involved in SSTR2 and DRD2 signal transduction. Finally, it is of interest to note that FLNA phosphorylation by PKA may be reduced by SSTR2 and DRD2, both negatively coupled to adenylyl cyclase. Since SSTR2 and DRD2 function/expression are regulated by FLNA itself, their ability to regulate FLNA phosphorylation might constitute a possible regulatory mechanism with still unknown consequences. These considerations suggest an interesting and unexplored link between camp/pka pathway and FLNA-mediated regulation of SS/DA receptors. Conclusions The recently revealed role of FLNA in DRD2 and SSTR2 receptors expression and signaling strongly suggests an association between FLNA expression and responsiveness of pituitary tumors to pharmacological therapy. FLNA can be phosphorylated by PKA on Ser2152, with increased FLNA resistance to cleavage by calpain, suggesting an involvement of camp signaling cascade, which in pituitary plays a major role in the regulation of cell proliferation and of hormone secretion, in the control of FLNA stability. Moreover, Ser2152 phosphorylation induces conformational changes affecting FLNA regions involved in SSTR2 and DRD2 binding and signal transduction, supporting the hypothesis of a camp-dependent regulation of FLNA ability to bind these receptors and to increase the efficiency of SS/DAmediated intracellular signal transduction by its scaffolding properties. Further studies are needed to understand the complex crosstalk between camp/pka pathway and FLNA-mediated regulation of SSTR2 and DRD2 receptors in pituitary. Acknowledgements This work was supported by an AIRC (Associazione Italiana Ricerca Cancro Milan) grant to G.M. (MFAG-8972) and by Ricerca Corrente Funds of Fondazione IRCCS Ca Granda-Milan. Conflict of Interest The authors declare that they have no conflicts of interest in the authorship or publication of this contribution. References 1 Ben-Jonathan N, Hnasko R. Dopamine as a prolactin (PRL) inhibitor. Endocr Rev 2001; 22: Patel YC. Somatostatin and its receptor family. Front Neuroendocrinol 1999; 20: Gueorguiev M, Grossman AB. Pituitary tumors in 2010: a new therapeutic era for pituitary tumors. Nat Rev Endocrinol 2011; 7: Ben-Shlomon A, Melmed S. Pituitary somatostatin receptor signaling. Trends Endocrinol Metab 2010; 21: Peverelli E, Lania AG, Mantovani G, Beck-Peccoz P, Spada A. Characterization of intracellular signaling mediated by human somatostatin receptor 5: role of the DRY motif and the third intracellular loop. Endocrinology 2009; 150: Peverelli E, Busnelli M, Vitali E, Giardino E, Galés C, Lania AG, Beck- Peccoz P, Chini B, Mantovani G, Spada A. Specific roles of G(i) protein family members revealed by dissecting SST5 coupling in human pituitary cells. J Cell Sci 2013; 126 (Pt 2): Ferrante E, Pellegrini C, Bondioni S, Peverelli E, Locatelli M, Gelmini P, Luciani P, Peri A, Mantovani G, Bosari S, Beck-Peccoz P, Spada A, Lania A. Octreotide promotes apoptosis in human somatotroph tumor cells by activating somatostatin receptor type 2. Endocr Relat Cancer 2006; 13: Sharma K, Patel YC, Srikant CB. Subtype-selective induction of wildtype p53 and apoptosis, but not cell cycle arrest, by human somatostatin receptor 3. Mol Endocrinol 1996; 10: Lamberts SW, de Herder WW, Hofland LJ. Somatostatin analogs in the diagnosis and treatment of cancer. Trends Endocrinol Metab 2002; 13: Colao A, Auriemma RS, Lombardi G, Pivonello R. Resistance to somatostatin analogs in acromegaly. Endocr Rev 2001; 32: Howlett TA 1, Willis D, Walker G, Wass JA, Trainer PJ. Control of growth hormone and IGF1 in patients with acromegaly in the UK: responses to medical treatment with somatostatin analogues and dopamine agonists. Clin Endocrinol (Oxf) 2013; 79: Molitch ME. Pharmacologic resistance in prolactinomas patients. Pituitary 2005; 8: Pellegrini I, Rasolonjanahary R, Gunz G, Bertrand P, Delivet S, Jedynak CP, Kordon C, Peillon F, Jaquet P, Enjalbert A. Resistance to bromocriptine in prolactinomas. J Clin Endocrinol Metab 1989; 69: Passos VQ, Fortes MAHZ, Giannella-Neto D, Bronstein MD. Genes differentially expressed in prolactinomas responsive and resistant to dopamine agonists. Neuroendocrinology 2009; 89: Caccavelli L, Feron F, Morange I, Rouer E, Benarous R, Dewailly D, Jaquet P, Kordon C, Enjalbert A. Decreased expression of the two D2 dopamine receptor isoforms in bromocriptine-resistant prolactinomas. Neuroendocrinology 1994; 60: Friedman E, Adams EF, Höög A, Gejman PV, Carson E, Larsson C, De Marco L, Werner S, Fahlbusch R, Nordenskjöld M. Normal structural dopamine type 2 receptor gene in prolactin-secreting and other pituitary tumors. J Clin Endocrinol Metab 1994; 78: Filopanti M., Barbieri AM, Angioni AR, Colao A, Gasco V, Grottoli S, Peri A, Baglioni S, Fustini MF, Pigliaru F, Monte PD, Borretta G, Ambrosi B, Jaffrain-Rea ML, Gasperi M, Brogioni S, Cannavò S, Mantovani G, Beck- Peccoz P, Lania A, Spada A. Dopamine D2 receptor gene polymorphisms and response to cabergoline therapy in patients with prolactin-secreting pituitary adenomas. Pharmacogenomics J 2008; 8: Reubi JC, Landolt AM. The growth hormone responses to octreotide in acromegaly correlate with tumor somatostatin receptor status. J Clin Endocrinol Metab 1989; 68: Plöckinger U, Albrecht S, Mawrin C, Saeger W, Buchfelder M, Petersenn S, Schulz S. Selective loss of somatostatin receptor 2 in octreotideresistant growth hormone-secreting tumors. J Clin Endocrinol Metab 2008; 93:

9 852 Review 20 Taboada GF, Luque RM, Neto LV, Machado Ede O, Sbaffi BC, Domingues RC, Marcondes JB, Chimelli LM, Fontes R, Niemeyer P, de Carvalho DP, Kineman RD, Gadelha MR. Quantitative analysis of somatostatin receptor subtypes (1 5) gene expression levels in somatotropinomas and correlation to in vivo hormonal and tumor volume responses to treatment with octreotide LAR. Eur J Endocrinol 2008; 158: Ferone D, de Herder WW, Pivonello R, Kros JM, van Koetsveld PM, de Jong T, Minuto F, Colao A, Lamberts SW, Hofland LJ. Correlation of in vitro and in vivo somatotropic tumor responsiveness to somatostatin analogs and dopamine agonists with immunohistochemical evaluation of somatostatin and dopamine receptors and electron microscopy. J Clin Endocrinol Metab 2008; 93: Colao A, Auriemma RS, Lombardi G, Pivonello R. Resistance to somatostatin analogs in acromegaly. Endocr Rev 2011; 32: Wildemberg LE, Neto LV, Costa DF, Nasciuti LE, Takiya CM, Alves LM, Rebora A, Minuto F, Ferone D, Gadelha MR. Low somatostatin receptor subtype 2, but not dopamine receptor subtype 2 expression predicts the lack of biochemical response of somatotropinomas to treatment with somatostatin analogs. J Endocrinol Invest 2013; 36: Bertherat J, Chanson P, Dewailly D, Enjalbert A, Jaquet P, Kordon C, Peillon F, Timsit J, Epelbaum J. Resistance to somatostatin (SRIH) analog therapy in acromegaly. Re-evaluation of the correlation between the SRIH receptor status of the pituitary tumor and the in vivo inhibition of GH secretion in response to SRIH analog. Horm Res 1992; 38: Bertherat J, Chanson P, Dewailly D, Dupuy M, Jaquet P, Peillon F, Epelbaum J. Somatostatin receptors, adenylate cyclase activity and growth hormone response to octreotide in GH-secreting tumors. J Clin Endocrinol Metab 1993; 7: Corbetta S, Ballare E, Mantovani G, Lania AG, Losa M, Di Blasio AM, Spada A. Somatostatin receptor subtype 2 and 5 in human GH-secreting pituitary tumors: analysis of gene sequence and mrna expression. Eur J Clin Invest 2001; 31: Petersenn S, Heyens M, Lüdecke DK, Beil FU, Schulte HM. Absence of somatostatin receptor type 2A mutations and gip oncogene in pituitary somatotroph tumors. Clin Endocrinol (Oxf) 2000; 52: Ballarè E, Persani L, Lania AG, Filopanti M, Giammona E, Corbetta S, Mantovani G, Arosio M, Beck-Peccoz P, Faglia G, Spada A. Mutation of somatostatin receptor type 5 in an acromegalic patient resistant to somatostatin analog treatment. J Clin Endocrinol Metab 2001; 86: Filopanti M, Ballare E, Lania AG, Bondioni S, Verga U, Locatelli M, Zavanone LM, Losa M, Gelmini S, Peri A, Orlando C, Beck-Peccoz P, Spada A. Loss of heterozygosity at the SS receptor type 5 locus in human GH- and TSH-secreting pituitary tumors. J Endocrinol Invest 2004; 27: Resmini E, Dadati P, Ravetti JL, Zona G, Spaziante R, Saveanu A, Jaquet P, Culler MD, Bianchi F, Rebora A, Minuto F, Ferone D. Rapid pituitary tumor shrinkage with dissociation between antiproliferative and antisecretory effects of a long-acting octreotide in an acromegalic patient. J Clin Endocrinol Metab 2007; 92: Daly AF, Tichomirowa MA, Petrossians P, Heliövaara E, Jaffrain-Rea ML, Barlier A, Naves LA, Ebeling T, Karhu A, Raappana A, Cazabat L, De Menis E, Montañana CF, Raverot G, Weil RJ, Sane T, Maiter D, Neggers S, Yaneva M, Tabarin A, Verrua E, Eloranta E, Murat A, Vierimaa O, Salmela PI, Emy P, Toledo RA, Sabaté MI, Villa C, Popelier M, Salvatori R, Jennings J, Longás AF, Aizpún JIL, Georgitsi M, Paschke R, Ronchi C, Valimaki M, Saloranta C, De Herder W, Cozzi R, Guitelman M, Magri F, Lagonigro MS, Halaby G, Corman V, Hagelstein M-T, Vanbellinghen J-F, Barra GB, Gimenez-Roqueplo A-P, Cameron FJ, Borson-Chazot F, Holdaway I, Toledo SPA, Stalla GK, Spada A, Zacharieva S, Bertherat J, Brue T, Bours V, Chanson P, Aaltonen LA, Beckers A. Clinical characteristics and therapeutic responses in patients with germ-line AIP mutations and pituitary adenomas: an international collaborative study. J Clin Endocrinol Metab 2010; 95: E373 E Stossel TP, Condeelis J, Cooley L, Hartwig JH, Noegel A, Schleicher M, Shapiro SS. Filamins as integrators of cell mechanics and signalling. Nature Rev Mol Cell Biol 2001; 2: van der Flier A, Sonnenberg A. Structural and functional aspects of filamins. Biochim Biophys Acta 2001; 1538: Hartwig JH, Stossel TP. Isolation and properties of actin, myosin, and a new actin binding protein in rabbit alveolar macrophages. J Biol Chem 1975; 250: Feng Y, Walsh CA. The many faces of filamin: a versatile molecular scaffold for cell motility and signalling. Nat Cell Biol 2004; 6: Fox JW, Lamperti ED, Ekşioğlu YZ, Hong SE, Feng Y, Graham DA, Scheffer IE, Dobyns WB, Hirsch BA, Radtke RA, Berkovic SF, Huttenlocher PR, Walsh CA. Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia. Neuron 1998; 21: Robertson SP 1, Twigg SR, Sutherland-Smith AJ, Biancalana V, Gorlin RJ, Horn D, Kenwrick SJ, Kim CA, Morava E, Newbury-Ecob R, Orstavik KH, Quarrell OW, Schwartz CE, Shears DJ, Suri M, Kendrick-Jones J, Wilkie AO. Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans. Nat Genet 2003; 33: Nakamura F, Stossel TP, Hartwig JH. The filamins: organizers of cell structure and function. Cell Adh Migr 2011; 5: Li M, Bermak JC, Wang ZW, Zhou QY. Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280). Mol Pharmacol 2000; 57: Lin R, Karpa K, Kabbani N, Goldman-Rakic P, Levenson R. Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A. Proc Natl Acad Sci USA 2001; 98: Lin R, Canfield V, Levenson R. Dominant negative mutants of filamin A block cell surface expression of the D2 dopamine receptor. Pharmacology 2002; 66: Peverelli E, Mantovani G, Vitali E, Elli FM, Olgiati L, Ferrero S, Laws ER, Della Mina P, Villa A, Beck-Peccoz P, Spada A, Lania AG. Filamin-A is essential for dopamine d2 receptor expression and signaling in tumorous lactotrophs. J Clin Endocrinol Metab 2012; 97: Scott MG, Pierotti V, Storez H, Lindberg E, Thuret A, Muntaner O, Labbé- Jullié C, Pitcher JA, Marullo S. Cooperative regulation of extracellular signal-regulated kinase activation and cell shape change by filamin A and beta-arrestins. Mol Cell Biol 2006; 26: Kim KM, Valenzano KJ, Robinson SR, Yao WD, Barak LS, Caron MG. Differential regulation of the dopamine D2 and D3 receptors by G protein-coupled receptor kinases and beta-arrestins. J Biol Chem 2001; 276: Zhang M, Breitwieser GE. High affinity interaction with filamin A protects against calcium-sensing receptor degradation. J Biol Chem 2005; 280: Seck T, Baron R, Horne WC. Binding of filamin to the C-terminal tail of the calcitonin receptor controls recycling. J Biol Chem 2003; 278: Thelin WR, Chen Y, Gentzsch M, Kreda SM, Sallee JL, Scarlett CO, Borchers CH, Jacobson K, Stutts MJ, Milgram SL. Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR. J Clin Invest 2007; 117: Beekman JM, van der Poel CE, van der Linden JA, van den Berg DL, van den Berghe PV, van de Winkel JG, Leusen JH. Filamin A stabilizes Fc gamma RI surface expression and prevents its lysosomal routing. J Immunol 2008; 180: Meller E, Puza T, Miller JC, Friedhoff AJ, Schweitzer JW. Receptor reserve for D2 dopaminergic inhibition of prolactin release in vivo and in vitro. J Pharmacol Exp Ther 1991; 257: Najib S 1, Saint-Laurent N, Estève JP, Schulz S, Boutet-Robinet E, Fourmy D, Lättig J, Mollereau C, Pyronnet S, Susini C, Bousquet C. A switch of G protein-coupled receptor binding preference from phosphoinositide 3-kinase (PI3K)-p85 to filamin A negatively controls the PI3K pathway. Mol Cell Biol 2012; 32: Peverelli E, Giardino E, Treppiedi D, Vitali E, Cambiaghi V, Locatelli M, Lasio G, Spada A, Lania A, Mantovani G. Filamin A (FLNA) plays an essential role in somatostatin receptor 2 (SST2) signaling and stabilization after agonist stimulation in human and rat somatotroph tumor cells. Endocrinology 2014 en PMID: Epub ahead of print 52 Stork PJ, Schmitt JM. Crosstalk between camp and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol 2002; 12: Peverelli E, Mantovani G, Lania AG, Spada A. camp in the pituitary: an old messenger for multiple signals. J Mol Endocrinol 2013; 52: R67 R77 54 Vallar L, Spada A, Giannattasio G. Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas. Nature 1987; 330: Landis CA, Masters SB, Spada A, Pace AM, Bourne HR, Vallar L. GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature 1989; 340: Lania A, Persani L, Ballaré E, Mantovani S, Losa M, Spada A. Constitutively active Gs alpha is associated with an increased phosphodiesterase activity in human growth hormone-secreting adenomas. J Clin Endocrinol Metab 1998; 83: Persani L, Borgato S, Lania A, Filopanti M, Mantovani G, Conti M, Spada A. Relevant camp-specific phosphodiesterase isoforms in human pituitary: effect of Gs(alpha) mutations. J Clin Endocrinol Metab 2001; 86:

10 Review Mantovani G, Bondioni S, Ferrero S, Gamba B, Ferrante E, Peverelli E, Corbetta S, Locatelli M, Rampini P, Beck-Peccoz P, Spada A, Lania AG. Effect of cyclic adenosine 3',5'-monophosphate/protein kinase a pathway on markers of cell proliferation in nonfunctioning pituitary adenomas. J Clin Endocrinol Metab 2005; 90: Vitali E, Peverelli E, Giardino E, Locatelli M, Lasio GB, Beck-Peccoz P, Spada A, Lania AG, Mantovani G. Cyclic adenosine 3' 5'-monophosphate (camp) exerts proliferative and anti-proliferative effects in pituitary cells of different types by activating both camp-dependent protein kinase A (PKA) and exchange proteins directly activated by camp (Epac). Mol Cell Endocrinol 2014; 383: Skalhegg BS, Tasken K. Specificity in the camp/pka signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci 2000; 5: D678 D Bertherat J, Chanson P, Montminy M. The cyclic adenosine 3,5 -monophosphate-responsive factor CREB is constitutively activated in human somatotroph adenomas. Mol Endocrinol 1995; 9: Wu MP, Jay D, Stracher A. Existence of multiple phosphorylated forms of human platelet actin binding protein. Cell Mol Biol Res 1994; 40: Chen M, Stracher A. In situ phosphorylation of platelet actin-binding protein by camp-dependent protein kinase stabilizes it against proteolysis by calpain. J Biol Chem 1989; 264: Zhang Z, Lawrence J, Stracher A. Phosphorylation of platelet actin binding protein protects against proteolysis by calcium dependent sulfhydryl protease. Biochem Biophys Res Commun 1988; 151: Jay D, García EJ, Lara JE, Medina MA, de la Luz Ibarra M. Determination of a camp-dependent protein kinase phosphorylation site in the C-terminal region of human endothelial actin-binding protein. Arch Biochem Biophys 2000; 377: Hammer A, Rider L, Oladimeji P, Cook L, Li Q, Mattingly RR, Diakonova M. Tyrosyl phosphorylated PAK1 regulates breast cancer cell motility in response to prolactin through filamin A. Mol Endocrinol 2013; 27: Cukier IH, Li Y, Lee JM. Cyclin B1/Cdk1 binds and phosphorylates Filamin A and regulates its ability to cross-link actin. FEBS Lett 2007; 581: Jay D, Stracher A. Expression in Escherichia coli and phosphorylation with camp-dependent protein kinase of the N-terminal region of human endothelial actin-binding protein. Biochem Biophys Res Commun 1994; 202: Jay D, García EJ, de la Luz Ibarra M. In situ determination of a PKA phosphorylation site in the C-terminal region of filamin. Mol Cell Biochem 2004; 260: Woo MS, Ohta Y, Rabinovitz I, Stossel TP, Blenis J. Ribosomal S6 kinase (RSK) regulates phosphorylation of filamin A on an important regulatory site. Mol Cell Biol 2004; 24: Zhang J, Neal J, Lian G, Shi B, Ferland RJ, Sheen V. Brefeldin A-inhibited guanine exchange factor 2 regulates filamin A phosphorylation and neuronal migration. J Neurosci 2012; 32: Eto A, Akita Y, Saido TC, Suzuki K, Kawashima S. The role of the calpaincalpastatin system in thyrotropin-releasing hormone-induced selective down-regulation of a protein kinase C isozyme, npkc epsilon, in rat pituitary GH4C1 cells. J Biol Chem 1995; 270: Sato-Kusubata K, Yajima Y, Kawashima S. Persistent activation of Gsalpha through limited proteolysis by calpain. Biochem J 2000; 347: Ohkawa K, Takada K, Asakura T, Hashizume Y, Okawa Y, Tashiro K, Ueda J, Itoh Y, Hibi N. Calpain inhibitor inhibits secretory granule maturation and secretion of GH. Neuroreport 2000; 11: Zheng X, Zhou AX, Rouhi P, Uramoto H, Borén J, Cao Y, Pereira T, Akyürek LM, Poellinger L. Hypoxia-induced and calpain-dependent cleavage of filamin A regulates the hypoxic response. Proc Natl Acad Sci USA 2014; 111: Ozanne DM, Brady ME, Cook S, Gaughan L, Neal DE, Robson CN. Androgen receptor nuclear translocation is facilitated by the f-actin crosslinking protein filamin. Mol Endocrinol 2000; 14: Loy CJ, Sim KS, Yong EL. Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions. Proc Natl Acad Sci USA 2003; 100: Wang Y, Kreisberg JI, Bedolla RG, Mikhailova M, devere White RW, Ghosh PM. A 90 kda fragment of filamin A promotes Casodex-induced growth inhibition in Casodex-resistant androgen receptor positive C4-2 prostate cancer cells. Oncogene 2000; 26: Xu Y, Bismar TA, Su J, Xu B, Kristiansen G, Varga Z, Teng L, Ingber DE, Mammoto A, Kumar R, Alaoui-Jamali MA. Filamin A regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion. J Exp Med 2010; 207: García E, Stracher A, Jay D. Calcineurin dephosphorylates the C-terminal region of filamin in an important regulatory site: a possible mechanism for filamin mobilization and cell signaling. Arch Biochem Biophys 2006; 446: Calderwood DA, Huttenlocher A, Kiosses WB, Rose DM, Woodside DG, Schwartz MA, Ginsberg MH. Increased filamin binding to beta-integrin cytoplasmic domains inhibits cell migration. Nat Cell Biol 2001; 3: Loo DT, Kanner SB, Aruffo A. Filamin binds to the cytoplasmic domain of the beta1-integrin. Identification of amino acids responsible for this interaction. J Biol Chem 1998; 273: Pfaff M, Liu S, Erle DJ, Ginsberg MH. Integrin beta cytoplasmic domains differentially bind to cytoskeletal proteins. J Biol Chem 1998; 273: Sharma CP, Ezzell RM, Arnaout MA. Direct interaction of filamin (ABP- 280) with the beta 2-integrin subunit CD18. J Immunol 1995; 154: Kiema T, Lad Y, Jiang P, Oxley CL, Baldassarre M, Wegener KL, Campbell ID, Ylänne J, Calderwood DA. The molecular basis of filamin binding to integrins and competition with talin. Mol Cell 2006; 21: Lad Y, Kiema T, Jiang P, Pentikäinen OT, Coles CH, Campbell ID, Calderwood DA, Ylänne J. Structure of three tandem filamin domains reveals auto-inhibition of ligand binding. EMBO J 2007; 26: Chen HS, Kolahi KS, Mofrad MR. Phosphorylation facilitates the integrin binding of filamin under force. Biophys J 2009; 97: Barlier A, Gunz G, Zamora AJ, Morange-Ramos I, Figarella-Branger D, Dufour H, Enjalbert A, Jaquet P. Pronostic and therapeutic consequences of Gs alpha mutations in somatotroph adenomas. J Clin Endocrinol Metab 1998; 83: Barlier A, Pellegrini-Bouiller I, Gunz G, Zamora AJ, Jaquet P, Enjalbert A. Impact of gsp oncogene on the expression of genes coding for Gsalpha, Pit-1, Gi2alpha, and somatostatin receptor 2 in human somatotroph adenomas: involvement in octreotide sensitivity. J Clin Endocrinol Metab 1999; 84: