Mechanisms Controlling the Activity of Localization ...

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Author's personal copy Mechanisms Controlling the Activity of Localization Signal Sequences Massimo D’Agostino, University of Lausanne, Epalinges, Switzerland Stefano Bonatti, University of Naples Federico II, Naples, Italy r 2017 Elsevier Inc. All rights reserved.

All eukaryotic cells contain numerous membrane-enclosed compartments, such as nucleus, endoplasmic reticulum (ER), Golgi complex, mitochondria, endosomes, lysosomes, and peroxisomes. The function of each compartment is assured by a specific set of proteins. Since protein synthesis occurs primarily in the cytosol, accurate translocation and protein trafficking from the cytosol to their final destinations is essential to maintain proper cellular function and activity. Indeed, it has been estimated that at least half of the intracellular proteins have to be transported to their functional destination (Chacinska et al., 2009). To be at the right place at the right time, proteins use specific localization signal sequences to interact with the molecular partners deputed to guarantee their correct localization. For many years the function of these localization signals was considered as a housekeeping function, but a number of studies have revealed that fine molecular mechanisms may control the activity of signal sequences. Interestingly, many proteins do not have a single subcellular localization but are present in more cellular compartments. For some of them, multiple localizations are due to the generation of different mRNA forms by alternative splicing. However, most of the time, proteins can occupy multiple cellular locations without any change in the amino acid sequence, indicating the existence of regulatory mechanisms based on post-translational events (Carrie and Whelan, 2013). These events include post-translational modifications and signal exposing/masking switching mechanisms that regulate the interaction between signals and transport proteins. A number of evidences showing how the function of signal sequences can be controlled is illustrated and discussed in this article.

Post-Translational Modifications Control Protein Localization One of the most common methods by which cells can control protein distribution is to modulate the binding affinity between the localization signals exposed by cargo proteins and their corresponding transporters. This modulation is usually controlled by posttranslational modifications that occur within or near the localization signals. This type of modification typically involves serine or threonine phosphorylation, lysine acetylation or sumoylation, and also myristoylation and palmitoylation. These modifications are thought either to interfere or to enhance the binding affinity of the signal sequences to their specific transporters. The most representative examples available are provided by the import/export mechanisms of nuclear proteins (Wilson and Dawson, 2011). Although small proteins can passively diffuse through nuclear pore complexes, the transport of many nuclear proteins carrying nuclear localization signals (NLSs) are mediated by import receptors called karyopherin or importins (Lange et al., 2007). In particular, the importin a/b mediate the nuclear import of proteins containing classical NLSs. The consensus patterns for the ̂ for class 2, KR-X-[W/F/Y]-X2-AF for class 3, [R/P]-X2-KR classical NLS are KR[K/R]R or K[K/R]RK for class 1, [P/R]-X2-KR[DE][K/R] ̂ [K/R][DE] for class 4, LGKR[K/R][W/F/Y] for class 5, and KR-X10-K12[K/R][K/R] or KR-X10-K12-[K/R]-X-[K/R] for the bipartite class, ̂ where [DE] represents any amino acids except aspartate and glutamate. On the other hand, only one class of nuclear export signals (NESs) is known and consists of a leucine rich short sequence (LR-NES). As it has been shown in a study of Cour et al. (2004) made on 67 NLSs, the consensus sequence is: [L/I/V/F/M]-X2,3-[L/IV/F/M]-X2,3-[L/I/V/F/M]-X-[L/I/V/F/M].

Phosphorylation-Dependent Inhibition of Localization Signal Recognition The nuclear import mechanism of many cell cycle-related proteins is controlled by phosphorylation events within or near their NLS that prevents binding to the transporter protein and thus suppresses nuclear import. For instance, the Saccharomyces cerevisiae transcription factors Swi5 is phosphorylated during cell cycle (late G1) by the cyclin-dependent kinase Cdc28 (homolog of mammalian CDK1) on serine residues within its NLS (636-KKYENVVIKRSPRKRGRPRKDGTSSVSSS-674), which reduces its nuclear import by suppressing the binding to its transporter (Moll et al., 1991; Sbia et al., 2008; Taberner et al., 2012). However, the cell cycle phosphatase Cdc14 reverts this phosphorylation during mitosis, reestablishing nuclear import (Visintin et al., 1998; Fig. 1(A)). In contrast, phosphorylation events within or near the NES have not been often reported. However, a good example is provided by the critical tumor suppressor p53 (Kracikova et al., 2013). As a consequence of ionizing radiation, ultraviolet light, nitric oxide, and reactive oxygen species (Liku et al., 2005), several DNA damage response kinases are activated: ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), protein kinase DNA-dependent catalytic polypeptide (PRKDC), and cyclin-dependent kinase 5 (CDK5) (Kadowaki et al., 2015). These kinases phosphorylate serine and threonine residues within the p53 NES motif (14-LSQETLSDLWKL-25), preventing the binding of exportin 1 to the NES and suppressing p53 nuclear export (Martinez et al., 1997; Banin et al., 1998; Canman et al., 1998; Tibbetts et al., 1999; Zhang and Xiong, 2001; Schneiderhan et al., 2003; Fig. 1(B)).

Phosphorylation-Dependent Promotion of Localization Signal Recognition Phosphorylation-based enhancement of localization signal recognition by transporter proteins is less frequent, but interesting examples have been reported as in the case of the mitogen-activated protein kinases 1 and 3 (MAPK1/ERK2/p38MAPK,

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Fig. 1 Phosphorylation-based inhibition of NLS/NES recognition. (A) Nuclear localization of Swi5 requires the exposure and recognition of a specific NLS whose activity is under the control of Cdc28 and Cdc14 kinases. Cdc28-dependent phosphorylation prevents NLS recognition, while Cdc14-dependent de-phosphorylation allows recognition by importin 7. Nuclear Swi5 induces the expression of several genes, including CDC6, MREI, ASHI, and EGT2. (B) Phosphorylation-dependent p53 nuclear export is controlled by several kinases, including ATM, ATR, PRKDC, and CDK5. Phosphorylation prevents NES recognition by exportin 1 and leads to p53 accumulation into the nucleus. ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3 related; CDK5, cyclin-dependent kinase 5; NES, nuclear export signal; NLS, nuclear localization signal; PRKDC, protein kinase DNA-dependent catalytic polypeptide.

MAPK3/ERK1/p42MAPK) that modulate growth factor signaling (Fig. 2(A)). When an extracellular growth factor is detected by a receptor tyrosine kinase that activates the MAPK pathway, the downstream MAPK kinase 1 (MAP2K1/MEK1) phosphorylates extracellular signal regulated kinase (ERK1/3) causing its release from a scaffold protein that keeps cytosolic ERK1/3 in its inactive

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Fig. 2 Phosphorylation-based promotion of NLS/NES recognition. (A) Phosphorylation-dependent nuclear localization of mitogen-activated protein kinases 1 and 3 (MAPK1/ERK2/p38MAPK, MAPK3/ERK1/p42MAPK). Upon activation of the MAPK pathway, MAPK kinase 1 (MEK1) phosphorylates ERK1/3, causing its release from a scaffold protein that keeps cytosolic ERK1/3 in its inactive state. Once released, ERK is recognized and phosphorylated by CK2 kinase on its NLS, rendering ERK recognizable by importin 7 and leading to nuclear import. (B) Phosphorylation-based export of androgen receptor (AR) from the nucleus is controlled by two kinases, ERK1/2 and JNK. Phosphorylation of AR occurs within its NES which is proximal to the DNA binding domain, thus causing AR release, recognition by exportin 1 and nuclear export. CK2, casein kinase 2; ERK, extracellular signal regulated kinase; JNK, c-Jun N-terminal kinase; NES, nuclear export signal; NLS, nuclear localization signal; PKC, protein kinase C; PM, plasma membrane.

state. Once released, ERK1/3 is recognized and phosphorylated by the casein kinase 2 (CK2) on its NLS. This second phosphorylation event renders ERK1/3 recognizable by importin 7 and leads to nuclear import (Chuderland et al., 2008; Zehorai et al., 2010). In addition, an example of phosphorylation-dependent nuclear export is provided by the androgen receptor (AR) which is phosphorylated on its NES at S650 by the stress-inducible c-Jun N-terminal kinase (JNK) or ERK1/3. This phosphorylation site is located within a NES proximal to the DNA binding domain. Thus, phosphorylation causes AR detachment from the DNA and enhances exportin 1 binding, leading to its export from the nucleus (Gioeli et al., 2006; Fig. 2(B)).

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Sumoylation and Acetylation-Based Control of Localization Signals Notably, there are only few experimental evidences that acetylation and/or sumoylation modulate localization signal recognition. One example of acetylation-dependent modulation of nuclear import is provided by RecQ protein-like 4 (RECQL4), a helicase involved in genomic stability. RECQL4 specifically interacts with the histone acetyltransferase p300 that acetylates one or more lysine residues at positions 376, 380, 382, 385, and 386 of RECQL4. These five lysine residues lie within a short motif (376-KQAWKQKWRKK-386) of 30 amino acids that is essential for the nuclear localization of RECQL4. Such acetylation suppresses NLS recognition and thus RECQL4 nuclear import (Dietschy et al., 2009; Fig. 3(A)). Also sumoylation may play a role in protein localization control. There is evidence that retinoic acid receptor alpha (RARA), which translocates into the nucleus to modulate gene transcription upon binding to all-trans retinoic acid (ATRA), is sumoylated near or within its NLS. In particular, the binding of ATRA induces a conformational change of RARA, revealing three lysine residues at positions 166, 171, and 399 that are sumoylated by a small ubiquitin-like modifier-2 (SUMO-2). The precise role of these modifications is still not known, but experimental evidences show that sumoylation of K399 leads to the recruitment of SUMO/ sentrin-specific peptidase 6 (SENP6 or Se6) that leads to desumoylate K171 and K166 before nuclear import of RARA, suggesting that a dynamic process of K166 and K171 sumoylation/desumoylation could influence the ATRA-controlled nuclear localization and transcriptional activity of RARA (Zhu et al., 2009; Fig. 3(B)). Moreover, another example is provided by the Krüppel-like factor

Fig. 3 Acetylation and sumoylation-based controls NLS activity. (A) Acetylation-based control of RecQ protein-like 4 (RECQL4) nuclear import. Histone acetyltransferase p300 poly-acetylates RECQL4 at residues close to the NLS. RECQL4 eventually accumulates in the cytosol because not recognizable by its importin. (B) Nuclear localization of retinoic acid receptor alpha (RARA) is controlled by sumoylation. Upon binding of all-trans retinoic acid (ATRA), RARA (rectangle) undergoes a conformational change (oval) that reveals K171, K166 and K399 residues for sumoylation (S) by SUMO-2. SUMO/sentrin-specific peptidase 6 (Se6) binds to the sumoylated K399 and de-sumoylates K171 and K166 residues before the complex may enter into the nucleus. NLS, nuclear localization signal; SUMO-2, sumoylated by a small ubiquitin-like modifier-2.

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5 (KLF5), a transcription factor involved in cell proliferation. KLF5 nuclear export is mediated by the exposure of a NES and its recognition by the nuclear export receptor CRM1. Upon sumoylation at lysine residues 151 and 202, the recognition of NES is suppressed, thereby increasing nuclear accumulation of KLF5 (Du et al., 2008).

Palmitoylation-Based Control of Protein Localization Palmitoylation is a reversible process, and several cellular proteins undergo dynamic palmitoylation (Smotrys and Linder, 2004; Salaun et al., 2010; Chamberlain et al., 2013; Frohlich et al., 2014; Dallavilla et al., 2016). It has been shown that this posttranslational modification provides an important mechanism for the regulation of protein subcellular localization, stability, trafficking, translocation to lipid rafts, aggregation, and interaction with effectors (Wright and Philips, 2006; Linder and Deschenes, 2007; Aicart-Ramos et al., 2011; Chamberlain et al., 2013; Frohlich et al., 2014; Dallavilla et al., 2016). One example regarding its role in subcellular localization control is provided by the dynamic palmitoylation pathway that regulates Ras trafficking (Goodwin et al., 2005). This dynamic palmitoylation results in a constant flux of the Ras proteins between Golgi complex and plasma membrane (Bleijlevens et al., 2008). In particular, palmitoylation at the Golgi membranes, mediated by palmitoyl-acyl-transferase (PAT), directs Ras to the PM via vesicle trafficking, whereas de-palmitoylation, mediated by acyl protein thioesterase (APT), allows the retrograde transport from the plasma membrane toward endosomal membranes and the Golgi apparatus, where Ras can be re-palmitoylated to start a new cycle (Bleijlevens et al., 2008; Fig. 4(A)). Moreover, palmitoylation was also observed for the synaptosomal-associated protein-25 (SNAP-25), a protein essential for neurotransmitter release from the synaptic terminals. SNAP-25 is synthesized as a soluble protein and palmitoylation on one or more cysteine residues drives its recruitment to membranes. The majority of cellular palmitoylation events are due to specific enzymes belonging to a family of 23 DHHC-domain containing proteins that function as palmitoyl-transferases (Fukata et al., 2004). SNAP-25 has two subcellular localization sites: Golgi complex and plasma membrane. The Golgi-localized DHHC3, DHHC7, and DHHC17 mediate Golgi localization, whereas DHHC2, which is closely related to DHHC15, mediates plasma membrane localization of SNAP-25 (Greaves et al., 2010; Fig. 4(B)). Thus, palmitoylation orchestrates the precise intracellular patterning of SNAP-25.

Localization Signal Controlled by Signal Exposing/Masking Mechanisms Protein localization is often linked to the exposure and recognition of specific signal sequences by transport proteins. Thus, a very efficient method to prevent protein transport is based on the physical burial of such sequences. Signals can be hidden either by a partner protein or a ligand, or by others copies of the same protein during the formation of multi-subunit complexes (intermolecular interaction); or by a conformational change of the protein (intramolecular interaction) (Neuberger et al., 2004; Hoelz et al., 2011). Moreover, when the signal is not constituted by a linear sequence of adjacent amino acid residues, conformational changes may drive the generation of tridimensional localization signal motif.

Intermolecular Mechanism The first well-characterized example of intermolecular mechanism leading to signal masking is provided by the T cell co-receptor CD8 (Hennecke and Cosson, 1993). CD8 is a transmembrane glycoprotein complex expressed in cytotoxic T lymphocytes and consists of two transmembrane subunits, a and b, that associate to form an heterodimer. Assembly of CD8b with CD8a is necessary for b subunit export from the ER and transport to the cell surface: CD8b chain, expressed in the absence of CD8a, forms a not functional homodimer that is retained in the ER. ER retention is typically mediated by arginine (RR, RXR, R-X2-R) or lysinebased signal sequences (KDEL, KKXX, or KXKXX) (Ellgaard and Helenius, 2003), whereas ER export is usually mediated by exposing, on the cytosolic domains, export signals that improve cargo loading into the COPII vesicles through the recruitment and binding of the Sec23-24 subunits of coatomer (Braulke and Bonifacino, 2009). Upon assembly with CD8a, the arginine-based retention signal present in the cytosolic domain of CD8b is masked. In addition, CD8a contains to its C-terminus a valine-based export signal (S-T/E-x-V), thus the heterodimeric complex is efficiently exported from the ER (D’Angelo et al., 2009; Lemma et al., 2013; D’Agostino et al., 2013, 2014; Fig. 5(A)). All together, this mechanism allows the cell to select the CD8 complex to expose on the plasma membrane (the not functional CD8bb homodimer is retained and degraded within the ER). A similar and well-characterized example is provided by the N-methyl-D-aspartic acid (NMDA) receptor (Hawkins et al., 2004). NMDA consists of two subunits, NR1 and NR2, that once synthesized are retained in the ER for the exposure of an arginine-based retention signals (RRR) present on both subunits. The formation of the properly assembled NMDA complex leads to the mutual masking of the ER retention signals. However, for an efficient ER export of the NMDA receptor, such masking mechanism has to be coupled to the exposure of an active ER export motif (HLFY) by the NR2 subunit (Hawkins et al., 2004). Thus, plasma membrane localization of functional NMDA receptors requires a complex interplay of retention signal masking and ER export signal exposure. An alternative and interesting mechanism is provided by the mini chromosome maintenance proteins (MCM2-7) that form the core of the replicative helicase whose activity is critical for the DNA replication origin (Onesti and Macneill, 2013). MCM2 and MCM3 have weak NLSs, but when in complex they form a single strong NLS and the other NESs present within MCM3 are masked and suppressed. Moreover, MCM4-7 subunits do not contain any NLS and their localization remains entirely dependent on the binding to MCM2 and MCM3 subunits (Dalton and Whitbread, 1995; Liku et al., 2005).

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Fig. 4 Palmitoylation controls protein localization. (A) Intracellular distribution of Ras protein is controlled by palmitoylation-dependent posttranslational modification. On the Golgi membranes, Ras is palmitoylated by palmitoyl-acyl-transferase (PAT) leading to its transport toward plasma membrane through anterograde vesicular carriers. At the plasma membrane, Ras de-palmitoylation mediated by acyl protein thioesterase (APT) allows its relocalization to the Golgi complex through retrograde vesicular carriers. (B) Synaptic vesicle release occurs at the presynaptic boutons of the terminal axons of neuronal cells. Upon palmitoylation mediated by DHHC2 protein, synaptosomal-associated protein-25 (SNAP-25, red) is recruited to the plasma membrane where it associates with its cognate SNAREs (blue and yellow) to drive membrane fusion and neurotransmitters release.

Intramolecular Interaction A well-characterized example of intramolecular masking is shown by the S. cerevisiae transcription factor Yap1, which mediates the oxidative stress response (Moye-Rowley et al., 1989). Under normal conditions, Yap1 shuttles between the nucleus and the

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Fig. 5 Masking mechanisms may control localization signal activity. (A) Intermolecular masking controls CD8 complex endoplasmic reticulum (ER) export. The exposure of an ER retention signal (RRR) restrains the homodimer CD8bb from being exported from the ER. The formation of the heterodimer CD8ab allows masks the ER retention signal and the heterodimer is exported as well as CD8aa homodimer thanks to the export signal present at the C-terminus of CD8a (ST/E-x-V). (B) Yap1 nuclear localization is controlled by oxidative-dependent conformational change. In the presence of an oxidative stress, Yap1 undergoes a conformational change due to the formation of a disulfide bond between two cysteine residues located at the C-terminal and core domain of the protein, respectively. Such a conformational change masks the NES but leaves active the NLS, thus leading to nuclear accumulation of Yap1. NES, nuclear export signal; NLS, nuclear localization signal.

cytoplasm via its NLS and NES located at the N- and C-termini of the protein, respectively (Wood et al., 2004). Oxidative stress induces the formation of an intramolecular disulfide bond between a cysteine near the NES and another cysteine on the protein backbone, thereby masking the NES (Yan et al., 1998; Wood et al., 2004; Okazaki et al., 2005; Rowe et al., 2008). Because the NES is masked under oxidative conditions, Yap1 exposes only the NLS and accumulates in the nucleus where activates the oxidative stress-response transcriptional program (Fig. 5(B)).

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A similar intramolecular mechanism for masking/exposing localization signals has been recently shown for the tumor suppressor protein PTEN that exerts its function at two subcellular locations, the plasma membrane and the nucleus, but is inactive when present in the cytosol (Nguyen et al., 2015). At the plasma membrane, PTEN controls cell proliferation and migration via the phosphoinositol PIP3; in the nucleus, it controls DNA repair and genome stability independently to PIP3. To be recruited at the PM, PTEN interacts with the lipid bilayer through its N-terminal PIP3-binding domain, its catalytic domain, and its C2 domain (core domains) (Das et al., 2003; Lumb and Sansom, 2013; Nguyen et al., 2014). More precisely, this recruitment is mediated by the interaction between positively charged residues in the PIP3-binding and C2 domains and the negatively charged membrane lipids. On the other hand, the cytosolic localization is ensured by the closed conformation of the protein, due to the phosphorylation of four serine/threonine residues (S380, T382, T383, and S385) in the tail domain. De-phosphorylation of the tail causes changes in the protein structure that switches to the opened conformation and exposes the membrane-binding regulatory motif in the core region, thus allowing the recruitment of PTEN to the plasma membrane. In addition, this conformational change allows the exposure of a lysine residue (L13) that, when ubiquitinated by the E3 ubiquitin ligase NEDD4-1, leads the transport of PTEN into the nucleus with a not yet clarified mechanism. Finally, another well-characterized example of intramolecular interaction is provided by the Bcl-2 family member Bax, a crucial mediator of apoptosis during development and disease (Schinzel et al., 2004). Activated Bax is tail-anchored in the mitochondrial outer membrane (MOM) through a C-terminal hydrophobic transmembrane domain (opened conformation)). In inactive Bax, this domain is folded back into a hydrophobic pocket (closed conformation) formed by its BH1, BH2 and BH3 domains (Suzuki et al., 2000). In this conformation, Bax resides in the cytosol in association with the 14-3-3 protein and an initial step is required to promote its activation and relocalization to the MOM (Capano and Crompton, 2002). It has been suggested that the N-terminal end of Bax controls its localization, since the removal of the first 19 N-terminal residues strongly stimulates Bax relocalization to the MOM (Cartron et al., 2003). In addition to the role of the N-terminal end, Bax activation and relocalization appears mainly dependent by the sequential phosphorylation of two C-terminal serine residues (S163 and S184) that promotes 14-3-3 dissociation and thus the switch to the opened conformation (Tsuruta et al., 2004). Finally, once localized to the mitochondrial membranes, Bax oligomerizes and forms pores that allow the release of cytochrome c and other apoptotic factors (Schinzel et al., 2004; Mandic et al., 2001).

Conformational-Based Generation of a Localization Signal Sequence Although the majority of localization signals are expressed as a linear amino acid sequence, some of them are formed upon protein conformational changes that align the amino acid residues on the surface of the protein. The advantage of this process is that conformational changes induced by allosteric events can transiently disrupt or reform the localization signal in response to the state of the protein. The disadvantage is that this mechanism renders almost impossible to predict the cellular localization from the primary amino acidic sequence. A well-characterized example is provided by the fatty acid-binding protein 5 (FABP5). When linoleic acid or arachidonic acid is bound to FABP5, some basic residues on the surface of the protein align each other (K24, R33, and K34). This alignment allows the binding to importin and transport of both FABP5 and its ligand into the nucleus. Conversely, when the ligand-binding pocket is emptied, the alignment of the basic residues is disrupted rendering the FABP5 not recognizable by importin and thereby preventing its nuclear import (Armstrong et al., 2014).

Conclusions Localization signal sequences play a pivotal role to guarantee the correct topological distribution of proteins in the cell. In many instances, the activity of these signals is controlled to promote or inhibit the final localization of the protein. Several molecular mechanisms are involved in such control and post-translational modifications and signal exposing/masking are the most frequently used. The post-translational modifications involved comprise serine/threonine phosphorylation, lysine acetylation or sumoylation, and also myristoylation and palmitoylation. These modifications either enhance or interfere with the binding affinity of the signal sequence to its specific transporter. Since these modifications are largely reversible, protein localization can be promoted or reverted by inducing or suppressing the modification, as nicely shown by the phosphorylation and de-phosphorylation events that control the localization of nuclear proteins among cytosol and nucleus. With this strategy, the cell controls more efficiently the activation or inactivation of several proteins needed to correctly execute specific processes. Moreover, this mechanism is frequently reinforced by the presence of interactor/chaperone proteins that help maintaining the inactive state. However, the activity of signal sequences can be also regulated by exposing/masking mechanisms that control either their activation or inhibition. These mechanisms are mediated by formation of protein complexes (intermolecular mechanism) or by conformational change of the protein itself (intramolecular mechanism), usually triggered by post-translational modifications. The first strategy is used by the cell to select and transport to the final destination only properly assembled and functional protein complexes. Interestingly, in the case of receptor protein complexes that must be exported from the ER to the cell surface, this mechanism is strictly coupled to the quality control processes in the ER (not correct complexes being degraded). The second strategy couples the advantages of reversible post-translational modifications (see above) with the strong effect that conformational changes may have on the exposing/masking of a signal sequence, thus better ensuring the distinction between the “on or off” state of the protein whose localization must be strictly controlled.

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The unifying conclusion that can be drawn by all the different examples illustrated in this article is that protein localization and relocalization is largely used by the cell as a tool to control when and where a protein has to exert its function.

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