Reversible phosphorylation of the signal transduction complex in Drosophila photoreceptors

In the Drosophila visual cascade, the transient receptor potential (TRP) calcium channel, phospholipase Cbeta (no-receptor-potential A), and an eye-specific isoform of protein kinase C (eye-PKC) comprise a multimolecular signaling complex via their interaction with the scaffold protein INAD. Previously, we showed that the interaction between INAD and eye-PKC is a prerequisite for deactivation of a light response, suggesting eye-PKC phosphorylates proteins in the complex. To identify substrates of eye-PKC, we immunoprecipitated the complex from head lysates using anti-INAD antibodies and performed in vitro kinase assays. Wild-type immunocomplexes incubated with [(32)P]ATP revealed phosphorylation of TRP and INAD. In contrast, immunocomplexes from inaC mutants missing eye-PKC, displayed no phosphorylation of TRP or INAD. We also investigated protein phosphatases that may be involved in the dephosphorylation of proteins in the complex. Dephosphorylation of TRP and INAD was partially suppressed by the protein phosphatase inhibitors okadaic acid, microcystin, and protein phosphatase inhibitor-2. These phosphatase activities were enriched in the cytosol of wild-type heads, but drastically reduced in extracts prepared from glass mutants, which lack photoreceptors. Our findings indicate that INAD functions as RACK (receptor for activated PKC), allowing eye-PKC to phosphorylate INAD and TRP. Furthermore, dephosphorylation of INAD and TRP is catalyzed by PP1/PP2A-like enzymes preferentially expressed in photoreceptor cells.     

Protein kinase C (PKC) isoforms in Drosophila

Six protein kinase C (PKC) genes are present in Drosophila, comprising two classical PKCs (PKC53E and eye-PKC), two novel PKCs (PKC98E and PKCdelta), an atypical PKC (DaPKC), and a PKC-related kinase. Loss of function alleles affecting DaPKC and eye-PKC are available and their mutant phenotypes have been characterized. DaPKC is essential for early embryonic development because it regulates cell polarity and asymmetric cell division. Eye-PKC plays a role in the regulation of visual signaling, a G-protein coupled phospholipase Cbeta-mediated cascade. Both eye-PKC and DaPKC are differentially localized through tethering to multimolecular complexes. DaPKC interacts with partitioning-defective 3 (Par-3) and Par-6 proteins, which contain PDZ (PSD95, DLG, ZO-1) domains. Similarly, eye-PKC is anchored to a PDZ domain containing scaffolding protein INAD. Characterization of these two PKCs in Drosophila revealed a universal mechanism by which PKC is tethered to specific protein complexes for participation in distinct signal transduction processes.     

The second PDZ domain of INAD is a type I domain involved in binding to eye protein kinase C. Mutational analysis and naturally occurring variants

INAD is a scaffolding protein containing five PSD95/dlg/zonular occludens-1 (PDZ) domains that tether NORPA (phospholipase Cbeta(4)), the TRP calcium channel, and eye-PKC in Drosophila photoreceptors. We previously showed that eye-PKC interacted with the second PDZ domain (PDZ2) of INAD. Sequence comparison with a prototypical type I PDZ domain predicts that PDZ2 is the best candidate among the five PDZ domains to recognize eye-PKC that contains a type I PDZ ligand, Ile-Thr-Ile-Ile, at its carboxyl terminus. Replacement of Ile(-3) in eye-PKC with charged residues resulted in a drastic reduction of the PDZ2 interaction. Substitution of a conserved His with Arg at the second alpha-helix of PDZ2 led to a reduced binding; however, a Leu replacement resulted in an enhanced eye-PKC association. We isolated and sequenced the InaD gene. The coding sequence of InaD contains nine exons spanning 3 kilobases. Translation of coding sequences from three wild-type alleles revealed three SNPs affecting residues, 282, 319, and 333 of INAD. These polymorphisms are localized in PDZ2. Interestingly, we found two of three PDZ2 variants displayed a greater affinity for eye-PKC. In summary, we evaluated the molecular basis of the eye-PKC and PDZ2 association by mutational analysis and concluded that PDZ2 of INAD is a type I domain important for the eye-PKC interaction.     

Coordination of an Array of Signaling Proteins through Homo- and Heteromeric Interactions Between PDZ Domains and Target Proteins

The rapid activation and feedback regulation of many G protein signaling cascades raises the possibility that the critical signaling proteins may be tightly coupled. Previous studies show that the PDZ domain containing protein INAD, which functions in Drosophila vision, coordinates a signaling complex by binding directly to the light-sensitive ion channel, TRP, and to phospholipase C (PLC). The INAD signaling complex also includes rhodopsin, protein kinase C (PKC), and calmodulin, though it is not known whether these proteins bind to INAD. In the current work, we show that rhodopsin, calmodulin, and PKC associate with the signaling complex by direct binding to INAD. We also found that a second ion channel, TRPL, bound to INAD. Thus, most of the proteins involved directly in phototransduction appear to bind to INAD. Furthermore, we found that INAD formed homopolymers and the homomultimerization occurred through two PDZ domains. Thus, we propose that the INAD supramolecular complex is a higher order signaling web consisting of an extended network of INAD molecules through which a G protein–coupled cascade is tethered.     

The visual G protein of fly photoreceptors interacts with the PDZ domain assembled INAD signaling complex via direct binding of activated Galpha(q) to phospholipase cbeta

Visual transduction in the compound eye of flies is a well-established model system for the study of G protein-coupled transduction pathways. Pivotal components of this signaling pathway, including the principal light-activated Ca(2+) channel transient receptor potential, an eye-specific protein kinase C, and the norpA-encoded phospholipase Cbeta, are assembled into a supramolecular signaling complex by the modular PDZ domain protein INAD. We have used immunoprecipitation assays to study the interaction of the heterotrimeric visual G protein with this INAD signaling complex. Light-activated Galpha(q)- guanosine 5'-O-(thiotriphosphate) and AlF(4)(-)-activated Galpha(q), but not Gbetagamma, form a stable complex with the INAD signaling complex. This interaction requires the presence of norpA-encoded phospholipase Cbeta, indicating that phospholipase Cbeta is the target of activated Galpha(q). Our data establish that the INAD signaling complex is a light-activated target of the phototransduction pathway, with Galpha(q) forming a molecular on-off switch that shuttles the visual signal from activated rhodopsin to INAD-linked phospholipase Cbeta.     

TRP and the PDZ protein, INAD, form the core complex required for retention of the signalplex in Drosophila photoreceptor cells

The light response in Drosophila photoreceptor cells is mediated by a series of proteins that assemble into a macromolecular complex referred to as the signalplex. The central player in the signalplex is inactivation no afterpotential D (INAD), a protein consisting of a tandem array of five PDZ domains. At least seven proteins bind INAD, including the transient receptor potential (TRP) channel, which depends on INAD for localization to the phototransducing organelle, the rhabdomere. However, the determinants required for localization of INAD are not known. In this work, we showed that INAD was required for retention rather than targeting of TRP to the rhabdomeres. In addition, we demonstrated that TRP bound to INAD through the COOH terminus, and this interaction was required for localization of INAD. Other proteins that depend on INAD for localization, phospholipase C and protein kinase C, also mislocalized. However, elimination of any other member of the signalplex had no impact on the spatial distribution of INAD. A direct interaction between TRP and INAD did not appear to have a role in the photoresponse independent of localization of multiple signaling components. Rather, the primary function of the TRP/ INAD complex is to form the core unit required for localization of the signalplex to the rhabdomeres.     

The organization of INAD-signaling complexes by a multivalent PDZ domain protein in Drosophila photoreceptor cells ensures sensitivity and speed of signaling

Phototransduction in Drosophila has emerged as an attractive model system for studying the organization of signaling cascades in vivo. In photoreceptor neurons, the multivalent PDZ protein INAD serves as a scaffold to assemble different components of the phototransduction pathway, including the effector PLC, the light-activated ion channel TRP, and a protein kinase C involved in deactivation of the light response. INAD is required for organizing and maintaining signaling complexes in the rhabdomeres of photoreceptors. This macromolecular organization endows photoreceptors with many of their signaling properties, including high sensitivity, fast activation and deactivation kinetics, and exquisite feedback regulation by small localized changes in [Ca2+]i. Assembly of transduction components into signaling complexes is also an important cellular strategy for ensuring specificity of signaling while minimizing unwanted cross-talk. In this report, we review INAD's role as a signal transduction scaffold and its role in the assembly and localization of photoreceptor complexes.     

Light-Dependent Phosphorylation of the Drosophila Inactivation No Afterpotential D (INAD) Scaffolding Protein at Thr170 and Ser174 by Eye-Specific Protein Kinase C

Drosophila inactivation no afterpotential D (INAD) is a PDZ domain-containing scaffolding protein that tethers components of the phototransduction cascade to form a supramolecular signaling complex. Here, we report the identification of eight INAD phosphorylation sites using a mass spectrometry approach. PDZ1, PDZ2, and PDZ4 each harbor one phosphorylation site, three phosphorylation sites are located in the linker region between PDZ1 and 2, one site is located between PDZ2 and PDZ3, and one site is located in the N-terminal region. Using a phosphospecific antibody, we found that INAD phosphorylated at Thr170/Ser174 was located within the rhabdomeres of the photoreceptor cells, suggesting that INAD becomes phosphorylated in this cellular compartment. INAD phosphorylation at Thr170/Ser174 depends on light, the phototransduction cascade, and on eye-Protein kinase C that is attached to INAD via one of its PDZ domains.     

Proteins modulating TRP channel function

TRP channels are involved in different signaling cascades; TRP channels can be activated via hormones and neurotransmitter in a receptor/G-protein-mediated manner or by osmotic, thermic or mechanic stimuli. The overall functional role of TRP channels within these processes of hormonal cellular control, nociception or cellular calcium homeostasis is still unclear, as these complex processes often involve macromolecular structures. Whereas the integration of Drosophila TRP in the phototransduction process is becoming clear, the understanding of the participation of mammalian TRP channels in signal transduction complexes is only beginning. TRP channels have been demonstrated to interact with PDZ domain proteins, and both scaffold and regulatory function have been shown for INAD, the PDZ domain protein of the Drosophila phototransduction complex. In mammalian cells, the interaction of NHERF and TRPC4 has been shown and it is anticipated that NHERF may abolish the apparent store-dependent regulation of TRPC4 and TRPC5. Whereas TRP channels and PDZ domain proteins form permanent heterodimeric proteins, the interaction of calcium-binding proteins is dependent on the calcium concentration and is, therefore, dynamic. The prototype of calcium-binding protein used for experiments is calmodulin; whether or not calmodulin is also the natural interaction partner of TRP channels is an open question.     

The roles of PDZ-containing proteins in PLC-beta-mediated signaling

Mammalian phospholipase C-beta isozymes are activated by a heterotrimeric GTP-binding protein linked to various cell surface receptors. Recent reports suggest that PDZ domain proteins play a significant role of PDZ-containing proteins in the regulation of mammalian PLC-beta isozymes. PDZ-containing proteins mediate the clustering of receptors and signaling molecules and thereby regulate agonist-induced signal transduction in polarized cells such as neuronal and epithelial cells. NORPA, a Drosophila PLC-beta, is known to be a component of a signaling complex that includes TRP and rhodopsin through interaction with INAD, a PDZ-containing protein. Mammalian PLC-beta1 and -beta2 isoforms interact with a PDZ-containing protein NHERF which is coupled to Trp4, a Ca(2+) channel. In addition, PLC-beta3 specifically interacts with E3KARP, another protein closely related to NHERF, through its C-terminal PDZ-binding motif. E3KARP up-regulates the PLC-beta3 activation coupled to muscarinic receptor. In this review, the role of signaling complexes mediated by PDZ-containing proteins in the regulation of PLC-beta isoforms will be discussed.     

The tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and inhibition of the Arf-GTPase-activating protein ASAP1

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.     

Identification of 14-3-3zeta as a protein kinase B/Akt substrate

Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.     

14-3-3 Protein mediates phosphorylation of microtubule-associated protein tau by serum- and glucocorticoid-induced protein kinase 1

The microtubule-associated protein, tau, is involved in numerous neuronal processes such as vesicle transport, microtubule-plasma membrane interaction and the intracellular localization of proteins. Tau is known to be phosphorylated by several kinases such as mitogen activated protein kinase, microtubule affinity regulating kinase, and protein kinase A. We found a putative serum- and glucocorticoid-induced protein kinase 1 (SGK1) phosphorylation site within the 207GSRSRTPSLP216 tau amino acid sequence. We report here that SGK1 phosphorylates Ser214 of Tau. Using a pull-down assay, we found that 14-3-3q interacts with SGK1 and tau to form a ternary protein complex that leads to phosphorylation of tau. 14-3-3 and phosphorylated tau were mainly co-localized in the nucleus of COS-1 cells. These results demonstrate that 14-3-3 scaffolds tau with SGK1 to facilitate the phosphorylation of tau at Ser214 and to regulate its subcellular localization.     

Regulation of a transient receptor potential (TRP) channel by tyrosine phosphorylation. SRC family kinase-dependent tyrosine phosphorylation of TRPV4 on TYR-253 mediates its response to hypotonic stress

The recently identified transient receptor potential (TRP) channel family member, TRPV4 (formerly known as OTRPC4, VR-OAC, TRP12, and VRL-2) is activated by hypotonicity. It is highly expressed in the kidney as well as blood-brain barrier-deficient hypothalamic nuclei responsible for systemic osmosensing. Apart from its gating by hypotonicity, little is known about TRPV4 regulation. We observed that hypotonic stress resulted in rapid tyrosine phosphorylation of TRPV4 in a heterologous expression model and in native murine distal convoluted tubule cells in culture. This tyrosine phosphorylation was sensitive to the inhibitor of Src family tyrosine kinases, PP1, in a dose-dependent fashion. TRPV4 associated with Src family kinases by co-immunoprecipitation studies and confocal immunofluorescence microscopy, and this interaction required an intact Src family kinase SH2 domain. One of these kinases, Lyn, was activated by hypotonic stress and phosphorylated TRPV4 in an immune complex kinase assay and an in vitro kinase assay using recombinant Lyn and TRPV4. Transfection of wild-type Lyn dramatically potentiated hypotonicity-dependent TRPV4 tyrosine phosphorylation whereas dominant negative-acting Lyn modestly inhibited it. Through mutagenesis studies, the site of tonicity-dependent tyrosine phosphorylation was mapped to Tyr-253, which is conserved across all species from which TRPV4 has been cloned. Importantly, point mutation of Tyr-253 abolished hypotonicity-dependent channel activity. In aggregate, these data indicate that hypotonic stress results in Src family tyrosine kinase-dependent tyrosine phosphorylation of the tonicity sensor TRPV4 at residue Tyr-253 and that this residue is essential for channel function in this context. This is the first example of direct regulation of TRP channel function through tyrosine phosphorylation.     

Endogenous phosphotyrosine signaling in zebrafish embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.     

Reversible tyrosine protein phosphorylation regulates large conductance voltage- and calcium-activated potassium channels via cortactin

Large conductance calcium- and voltage-activated potassium (BK) channels assemble as macromolecular signaling complexes and are potently regulated by reversible protein phosphorylation. However, although numerous studies have revealed regulation of BK channels through changes in direct phosphorylation of the pore-forming alpha-subunits the functional role of changes in phosphorylation of defined adapter/signaling proteins within the complex on channel function are essentially not known. Here, we demonstrate that mammalian BK channels are potently regulated by endogenous protein-tyrosine kinase and protein-tyrosine phosphatase activity closely associated with the channel. BK channel regulation was not dependent upon direct phosphorylation of the BK alpha-subunit, rather channel function was controlled by the tyrosine phosphorylation status of the adapter protein cortactin that assembles directly with the BK channel. Our data thus reveal a novel mode for BK channel regulation by reversible tyrosine phosphorylation and strongly support the hypothesis that phosphorylation-dependent regulation of accessory proteins within the BK channel signaling complex represents an important target for control of BK channel function.     

Dynein light chain LC8 negatively regulates NF-kappaB through the redox-dependent interaction with IkappaBalpha

Redox regulation of nuclear factor kappaB (NF-kappaB) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation, and we identified the dynein light chain LC8, which interacts with the NF-kappaB inhibitor IkappaBalpha, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-kappaB activation is redox-dependently regulated through LC8. LC8 inhibited TNFalpha-induced NF-kappaB activation in HeLa cells by interacting with IkappaBalpha and thereby preventing its phosphorylation by IkappaB kinase (IKK), without affecting the activity of IKK itself. TNFalpha induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys(2) residues of each subunit and thereby resulted in its dissociation from IkappaBalpha. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IkappaBalpha by TNFalpha stimulation. In addition LC8 inhibited NF-kappaB activation by other stimuli including interleukin-1beta and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IkappaBalpha in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.     

Dissecting independent channel and scaffolding roles of the Drosophila transient receptor potential channel

Drosophila transient receptor potential (TRP) serves dual roles as a cation channel and as a molecular anchor for the PDZ protein, INAD (inactivation no afterpotential D). Null mutations in trp cause impairment of visual transduction, mislocalization of INAD, and retinal degeneration. However, the impact of specifically altering TRP channel function is not known because existing loss-of-function alleles greatly reduce protein expression. In the current study we describe the isolation of a set of new trp alleles, including trp(14) with an amino acid substitution juxtaposed to the TRP domain. The trp(14) flies stably express TRP and display normal molecular anchoring, but defective channel function. Elimination of the anchoring function alone in trp(Delta)(1272), had minor effects on retinal morphology whereas disruption of channel function caused profound light-induced cell death. This retinal degeneration was greatly suppressed by elimination of the Na(+)/Ca(2+) exchanger, CalX, indicating that the cell death was due primarily to deficient Ca(2+) entry rather than disruption of the TRP-anchoring function.