Differential substrate specificity of ERK, JNK, and p38 MAP kinases toward Connexin 43.

Phosphorylation of connexin 43 (Cx43) is an important regulatory mechanism of gap junction (GJ) function. Cx43 is modified by several kinases on over 15 sites within its ∼140 amino acid-long C-terminus (CT). Phosphorylation of Cx43CT on S255, S262, S279, and S282 by ERK has been widely documented in several cell lines, by many investigators. Phosphorylation of these sites by JNK and p38, on the other hand, is not well-established. Indeed, ERK is a kinase activated by growth factors and is upregulated in diseases, such as cancer. JNK and p38, however, have a largely tumor-suppressive function due to their stress-activated and apoptotic role. We investigated substrate specificity of all three MAPKs toward Cx43CT, both in vitro and in two cell lines (MDCK - non-cancerous, epithelial cells and porcine PAECs - pulmonary artery endothelial cells). Cx43 phosphorylation was monitored through gel-shift assays on an SDS-PAGE, immunodetection with phospho-Cx43 antibodies, and LC-MS/MS phosphoproteomic analyses. Our results demonstrate that p38 and JNK specificity differ from each other and from ERK. JNK has a strong preference for S255 and S279, while p38 readily phosphorylates S279 and S282. In addition, while we confirmed that ERK can phosphorylate all four serines (255, 262, 279, and 282), we identified T290 as a novel ERK phosphorylation site. This work underscores the importance of delineating the effects of ERK, JNK, and p38 signaling pathways on Cx43 and GJ function.

Inhibiting Epidermal Growth Factor Receptor Dimerization and Signaling Through Targeted Delivery of a Juxtamembrane Domain Peptide Mimic.

Overexpression and deregulation of the epidermal growth factor receptor (EGFR) are implicated in multiple human cancers and therefore are a focus for the development of therapeutics. Current strategies aimed at inhibiting EGFR activity include monoclonal antibodies and tyrosine kinase inhibitors. However, activating mutations severely limit the efficacy of these therapeutics. There is thus a growing need for novel methods to inhibit EGFR. One promising approach involves blocking the association of the cytoplasmic juxtamembrane (JM) domain of EGFR, which has been shown to be essential for receptor dimerization and kinase function. Here, we aim to improve the selectivity and efficacy of an EGFR JM peptide mimic by utilizing the pH(low) insertion peptide (pHLIP), a unique molecule that can selectively target cancer cells solely based on their extracellular acidity. This delivery strategy potentially allows for more selective targeting to tumors than current methods and for anchoring the peptide mimic to the cytoplasmic leaflet of the plasma membrane, increasing its local concentration and thus efficacy. We show that the conjugated construct is capable of inhibiting EGFR phosphorylation and downstream signaling and of inducing concentration- and pH-dependent toxicity in cervical cancer cells. We envision that this approach could be expanded to the modulation of other single-span membrane receptors whose activity is mediated by JM domains.

Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover.

To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin-Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.

Oligomeric state regulated trafficking of human platelet-activating factor acetylhydrolase type-II.

The intracellular enzyme platelet-activating factor acetylhydrolase type-II (PAFAH-II) hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. PAFAH-II in its resting state is mainly cytoplasmic, and it responds to oxidative stress by becoming increasingly bound to endoplasmic reticulum and Golgi membranes. Numerous studies have indicated that this enzyme is essential for protecting cells from oxidative stress induced apoptosis. However, the regulatory mechanism of the oxidative stress response by PAFAH-II has not been fully resolved. Here, changes to the oligomeric state of human PAFAH-II were investigated as a potential regulatory mechanism toward enzyme trafficking. Native PAGE analysis in vitro and photon counting histogram within live cells showed that PAFAH-II is both monomeric and dimeric. A Gly-2-Ala site-directed mutation of PAFAH-II demonstrated that the N-terminal myristoyl group is required for homodimerization. Additionally, the distribution of oligomeric PAFAH-II is distinct within the cell; homodimers of PAFAH-II were localized to the cytoplasm while monomers were associated to the membranes of the endoplasmic reticulum and Golgi. We propose that the oligomeric state of PAFAH-II drives functional protein trafficking. PAFAH-II localization to the membrane is critical for substrate acquisition and effective oxidative stress protection. It is hypothesized that the balance between monomer and dimer serves as a regulatory mechanism of a PAFAH-II oxidative stress response.

Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity.

Eukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the "DFG+1" residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.

Proteins and mechanisms regulating gap-junction assembly, internalization, and degradation.

Gap junctions (GJs) are the only known cellular structures that allow a direct cell-to-cell transfer of signaling molecules by forming densely packed arrays or "plaques" of hydrophilic channels that bridge the apposing membranes of neighboring cells. The crucial role of GJ-mediated intercellular communication (GJIC) for all aspects of multicellular life, including coordination of development, tissue function, and cell homeostasis, has been well documented. Assembly and degradation of these membrane channels is a complex process that includes biosynthesis of the connexin (Cx) subunit proteins (innexins in invertebrates) on endoplasmic reticulum (ER) membranes, oligomerization of compatible subunits into hexameric hemichannels (connexons), delivery of the connexons to the plasma membrane (PM), head-on docking of compatible connexons in the extracellular space at distinct locations, arrangement of channels into dynamic spatially and temporally organized GJ channel plaques, as well as internalization of GJs into the cytoplasm followed by their degradation. Clearly, precise modulation of GJIC, biosynthesis, and degradation are crucial for accurate function, and much research currently addresses how these fundamental processes are regulated. Here, we review posttranslational protein modifications (e.g., phosphorylation and ubiquitination) and the binding of protein partners (e.g., the scaffolding protein ZO-1) known to regulate GJ biosynthesis, internalization, and degradation. We also look closely at the atomic resolution structure of a GJ channel, since the structure harbors vital cues relevant to GJ biosynthesis and turnover.

Degradation of endocytosed gap junctions by autophagosomal and endo-/lysosomal pathways: a perspective.

Gap junctions (GJs) are composed of tens to many thousands of double-membrane spanning GJ channels that cluster together to form densely packed channel arrays (termed GJ plaques) in apposing plasma membranes of neighboring cells. In addition to providing direct intercellular communication (GJIC, their hallmark function), GJs, based on their characteristic double-membrane-spanning configuration, likely also significantly contribute to physical cell-to-cell adhesion. Clearly, modulation (up-/down-regulation) of GJIC and of physical cell-to-cell adhesion is as vitally important as the basic ability of GJ formation itself. Others and we have previously described that GJs can be removed from the plasma membrane via the internalization of entire GJ plaques (or portions thereof) in a cellular process that resembles clathrin-mediated endocytosis. GJ endocytosis results in the formation of double-membrane vesicles [termed annular gap junctions (AGJs) or connexosomes] in the cytoplasm of one of the coupled cells. Four recent independent studies, consistent with earlier ultrastructural analyses, demonstrate the degradation of endocytosed AGJ vesicles via autophagy. However, in TPA-treated cells others report degradation of AGJs via the endo-/lysosomal degradation pathway. Here we summarize evidence that supports the concept that autophagy serves as the cellular default pathway for the degradation of internalized GJs. Furthermore, we highlight and discuss structural criteria that seem required for an alternate degradation via the endo-/lysosomal pathway.

Trafficking of platelet-activating factor acetylhydrolase type II in response to oxidative stress.

Platelet-activating factor acetylhydrolase type II (PAFAH-II) is an intracellular phospholipase A(2) enzyme that hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. This N-terminally myristoylated protein becomes associated with cytoplasm-facing cell membranes under oxidative stress. The structural requirements for binding of PAFAH-II to membranes in response to oxidative stress are unknown. To begin elucidating the mechanism of trafficking and stress response, we constructed a homology model of PAFAH-II. From the predicted membrane orientation of PAFAH-II, the N-terminal myristoyl group and a hydrophobic patch are hypothesized to be involved in membrane binding. Localization studies of human PAFAH-II in HEK293 cells indicated that an unmyristoylated mutant remained cytoplasmic under stressed and unstressed conditions. The myristoylated wild-type enzyme was partially localized to the cytoplasmic membranes prior to stress and became more localized to these membranes upon stress. A triple mutation of three hydrophobic patch residues of the membrane binding region likewise did not localize to membranes following stress. These results indicate that both the myristoyl group and the hydrophobic patch are essential for proper trafficking of the enzyme to the membranes following oxidative stress. Additionally, colocalization studies using organelle-specific proteins demonstrate that PAFAH-II is transported to the membranes of both the endoplasmic reticulum and Golgi apparatus.

GST pi modulates JNK activity through a direct interaction with JNK substrate, ATF2.

Human GSTpi, an important detoxification enzyme, has been shown to modulate the activity of JNKs by inhibiting apoptosis and by causing cell proliferation and tumor growth. In this work, we describe a detailed analysis of the interaction in vitro between GSTpi and JNK isoforms (both in their inactive and active, phosphorylated forms). The ability of active JNK1 or JNK2 to phosphorylate their substrate, ATF2, is inhibited by two naturally occurring GSTpi haplotypes (Ile105/Ala114, WT or haplotype A, and Val105/Val114, haplotype C). Haplotype C of GSTpi is a more potent inhibitor of JNK activity than haplotype A, yielding 75-80% and 25-45% inhibition, respectively. We show that GSTpi is not a substrate of JNK, as was earlier suggested by others. Through binding studies, we demonstrate that the interaction between GSTpi and phosphorylated, active JNKs is isoform specific, with JNK1 being the preferred isoform. In contrast, GSTpi does not interact with unphosphorylated, inactive JNKs unless a JNK substrate, ATF2, is present. We also demonstrate, for the first time, a direct interaction: between GSTpi and ATF2. GSTpi binds with similar affinity to active JNK + ATF2 and to ATF2 alone. Direct binding experiments between ATF2 and GSTpi, either alone or in the presence of glutathione analogs or phosphorylated ATF2, indicate that the xenobiotic portion of the GSTpi active site and the JNK binding domain of ATF2 are involved in this interaction. Competition between GSTpi and active JNK for the substrate ATF2 may be responsible for the inhibition of JNK catalysis by GSTpi.

Activation by phosphorylation and purification of human c-Jun N-terminal kinase (JNK) isoforms in milligram amounts.

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) signaling cascade. They are activated through dual phosphorylation of two residues in the activation loop, a threonine and a tyrosine, by MAP2 kinases (MKK4 and 7) in response to various extracellular stresses such as UV or osmotic shock, as well as by cytokines and growth factors. Only small amounts of phosphorylated, active JNKs have previously been produced because of difficulties in expressing these phosphorylated kinases in Escherichia coli, which lack the appropriate upstream kinases. We have now established a novel activation and purification method that allows for reproducible production of milligram amounts of active, phosphorylated JNKs suitable for a variety of enzymatic, biophysical and structural characterizations. We utilize N-terminally His-tagged MKK4 that is coexpressed in E. coli with a constitutively active form of MEKK1. This phosphorylated, active His-MKK4 is purified by Ni-NTA chromatography and used to phosphorylate milligram amounts of three different isoforms of human JNKs (JNK1α1, JNK1α2 and JNK2α2) that had separately been expressed and purified from E. coli in their inactive forms. These in vitro activated JNKs are phosphorylated on both residues (T183, Y185) in their activation loops and are active towards their substrate, ATF2.