Kinase-like Domain Research Articles

Liprin-Mediated Large Signaling Complex Organization Revealed by the Liprin-α/CASK and Liprin-α/Liprin-β Complex Structures

Liprins are highly conserved scaffold proteins that regulate cell adhesion, cell migration, and synapse development by binding to diverse target proteins. The molecular basis governing liprin/target interactions is poorly understood. The liprin-α2/CASK complex structure solved here reveals that the three SAM domains of liprin-α form an integrated supramodule that binds to the CASK kinase-like domain. As supported by biochemical and cellular studies, the interaction between liprin-α and CASK is unique to vertebrates, implying that the liprin-α/CASK interaction islikely to regulate higher-order brain functions in mammals. Consistently, we demonstrate that three recently identified X-linked mental retardation mutants of CASK are defective in binding to liprin-α. We also solved the liprin-α/liprin-β SAM domain complex structure, which uncovers the mechanism underlying liprin heterodimerizaion. Finally, formation of the CASK/liprin-α/liprin-β ternary complex suggests that liprins can mediate assembly of target proteins into large protein complexes capable of regulating numerous cellular activities.

MTOR and the differentiation of mesenchymal stem cells

The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine protein kinase, belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family, which contains a lipid kinase-like domain within their C-terminal region. Recent studies have revealed that mTOR as a critical intracellular molecule can sense the extracellular energy status and regulate the cell growth and proliferation in a variety of cells and tissues. This review summarizes our current understanding about the effects of mTOR on cell differentiation and tissue development, with an emphasis on the lineage determination of mesenchymal stem cells. mTOR can promote adipogenesis in white adipocytes, brown adipocytes, and muscle satellite cells, while rapamycin inhibits the adipogenic function of mTOR. mTOR signaling may function to affect osteoblast proliferation and differentiation, however, rapamycin has been reported to either inhibit or promote osteogenesis. Although the precise mechanism remains unclear, mTOR is indispensable for myogenesis. Depending on the cell type, rapamycin has been reported to inhibit, promote, or have no effect on myogenesis.

Inhibition of RNase L and RNA-dependent Protein Kinase (PKR) by Sunitinib Impairs Antiviral Innate Immunity

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 μM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-β levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.

Knockdown of the Zebrafish Ortholog of the Retinitis Pigmentosa 2 (RP2) Gene Results in Retinal Degeneration

The authors investigated the expression and function of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene. Zebrafish RP2 (ZFRP2) cDNA was isolated from adult eye mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Gene expression was examined by RT-PCR. The deduced peptide sequence was aligned with RP2 orthologues from different species. Translational suppression (knockdown) of zebrafish RP2 was carried out by antisense morpholino-injection. The phenotype of ZFRP2 knockdown morphants was characterized by immunohistology and histology. Human wild-type and mutant RP2 mRNAs were coinjected with ZFRP2 morpholinos to test whether human RP2 mRNA could rescue ZFRP2 knockdown phenotypes. ZFRP2 encodes a protein of 376 amino acids containing an N-terminal tubulin folding cofactor C-like domain and a C-terminal nucleoside diphosphate kinase-like domain. It shares 63% to 65% amino acid identity with human, mouse and bovine RP2. RP2 is expressed at the earliest stages of zebrafish development and persists into adulthood. Knockdown of RP2 in zebrafish causes a curved body axis and small eye phenotype, associated with increased cell death throughout the retina. Human wild-type RP2 mRNA could rescue the body curvature phenotype of ZFRP2 morphants, and the eye size of the resultant morphants was significantly increased over that of morphants in which ZFRP2 had been depleted. Zebrafish RP2 is widely expressed throughout development. ZFRP2 knockdown caused retinal degeneration in zebrafish. Human RP2 could partially rescue the small eye phenotype of ZFRP2 morphants.

Abstract 3131: A novel tyrosine kinase signaling pathway in human basal breast cancer

Abstract Basal-type breast cancers account for up to 25 % of breast cancer cases, depending on the patient population. These cancers usually lack estrogen, progesterone and erbB2 receptors and are therefore resistant to endocrine or trastuzumab therapy, and markers that stratify this subset according to clinical behaviour have yet to be identified. Consequently, basal breast cancers present a major treatment dilemma. In order to identify potential therapeutic targets and prognostic markers for this disease subgroup, we are currently undertaking a detailed characterization of tyrosine kinase signaling networks in basal breast cancer cells. Mass spectrometry (MS)-based global profiling of the tyrosine phosphoproteome of basal breast cancer cells identified a prominent Src family kinase (SFK) signaling network that featured high expression of the SFK Lyn (Hochgräfe et al, Cancer Res 2010 doi: 10.1158/0008-5472.CAN-10-0911). In order to identify Lyn substrates, we used stable isotope labelling with amino acids in cell culture (SILAC) in combination with immunoaffinity-based phosphotyrosine enrichment and LC-MS/MS to profile cellular tyrosine phosphorylation following transient siRNA-mediated Lyn knockdown in MDA-MB-231 and BT-549 basal breast cancer cells. This identified several Lyn substrates characteristic of both cell lines, which included annexin A2 (Y42), 40S ribosomal protein S10 (Y12) and SgK269 (Y635). The latter is a relatively understudied kinase that contains several tyrosine phosphorylation sites that are likely to act as recruitment sites for SH2/PTB domain-containing signalling proteins, and a C-terminal kinase-like domain that lacks the consensus DFG motif usually regarded as essential for kinase activity. Protein expression of SgK269 was markedly higher in basal, versus luminal, breast cancer cells, while mRNA transcript levels in the two cell types were similar, indicating post-transcriptional regulation of SgK269 expression. Phosphorylation of SgK269 on Y635 could be detected readily in basal breast cancer cells. Overexpression of SgK269 in MCF-10A immortalized mammary epithelial cells led to a more elongated morphology in monolayer and perturbed acinar morphogenesis in Matrigel, resulting in large, multilobular acini that failed to undergo proliferative suppression in long-term culture. SgK269-overexpressing cells also exhibited enhanced tyrosine phosphorylation of Stat3. In addition, we undertook stable shRNA-mediated knockdown of SgK269 in BT-549 cells. This reduced soft agar growth and Stat3 activation. These studies identify a novel oncogenic signalling pathway in basal breast cancer involving the kinases Lyn and SgK269, which may represent a target for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3131. doi:10.1158/1538-7445.AM2011-3131

Organization of PDZ Domains in Full-Length PSD-95

PDZ (PSD-95/Dlg/ZO-1) domains are small (∼90 amino acids) protein-interacting modules that canonically bind c-terminal recognition motifs. Multiple copies of PDZ domains within a single protein sequence are often required for the proper functioning of multidomain scaffold proteins. While the binding properties of isolated domains have been relatively well characterized, recent studies have demonstrated that interactions between PDZ domains are capable of modulating the binding affinities of individual domains. Interdomain association between PDZ domains may play a crucial role in the proper functioning of PDZ containing proteins but to date has been left largely uninvestigated. PSD-95 is the canonical member of the membrane associated guanylate kinase (MAGUK) family of PDZ containing proteins. Like all MAGUKs, PSD-95 is composed of three sequential PDZ domains followed by an SH3 domain and an enzymatically inactive guanylate kinase-like domain. The flexible linker regions between each PDZ domain and the apparent lack of a fixed domain organization have made it difficult to characterize these domains at the bulk ensemble level. Single-molecule FRET (smFRET) is a structural biology tool that is unaffected by the presence or absence of protein structures outside the regions of interest, and is capable of accurately measuring long range distances in dynamically fluctuating samples without the averaging effects inherent in ensemble measurements. smFRET measurements between multiple sites on each PDZ domain were used to observe the position and orientation of PDZ domains in both full-length PSD-95 as well as in truncated fragments. In combination with fluorescence correlation spectroscopy, isothermal titration calorimetry and molecular simulations, smFRET was used to determine the dynamic structure of PDZ domains in PSD-95 and to determine how this structure influences the binding modes for physiological ligands.

Human Tribbles homolog 1 functions as a negative regulator of retinoic acid receptor

Tribbles encode an evolutionarily conserved protein family that regulates cell proliferation, motility, metabolism and oncogenic transformation. Emerging evidence suggests that Tribbles function as adaptor or scaffold proteins to facilitate the degradation of their target proteins and to control the activation of various key signaling pathways. In this study, we uncover a novel function of human Tribbles homolog 1 (Trib1) as a regulator of retinoic acid receptor (RAR) signaling. We show that shRNA-mediated knockdown of Trib1 promotes transcriptional activity of RARs, leading to enhanced expression of endogenous RAR-target genes. Moreover, our results show that Trib1 directly interacts with RARα and retinoid X receptor-α (RXRα) through its kinase-like domain. Consistently, Trib1 colocalizes with RARα and RXRα in the nucleus. Biochemical analyses show that the ligand-binding domain (LBD) of RARα mediates the interaction with Trib1. Ligand treatment, however, does not affect the binding of Trib1 to RARα/RXRα. Furthermore, a putative LXXLL motif, which is a potential LBD-binding site and locates in the kinase-like domain of Trib1, is not required for the binding.These results suggest a unique feature of the binding. Taken together, these results suggest that Trib1 functions as a negative regulator of RARs and shed new light on the molecular mechanisms for nuclear receptor-mediated transcriptional repression.

SS6-2 Inhibition of RNAse L and PKR by the anti-cancer drug sunitinib impairs innate immunity in virus-infected mice

Kinases control virtually all aspects of biology. Forty-eight human proteins have a kinase-like domain that lacks at least one of the conserved catalytic residues; these proteins are therefore predicted to be inactive and have been termed pseudokinases. Here, we describe exciting work suggesting that pseudokinases, despite lacking the ability to phosphorylate substrates, are still pivotal in regulating diverse cellular processes. We review evidence that the pseudokinase STRAD controls the function of the tumour suppressor kinase LKB1 and that a single amino acid substitution within the pseudokinase domain of the tyrosine kinase JAK2 leads to several malignant myeloproliferative disorders. We also discuss the emerging functions of other pseudokinases, including HER3 (also called ErbB3), EphB6, CCK4 (also called PTK7), KSR, Trb3, GCN2, TRRAP, ILK and CASK.

Oligomerization of Cavβ subunits is an essential correlate of Ca2+channel activity

Voltage-gated calcium channels conduct Ca(2+) ions in response to membrane depolarization. The resulting transient increase in cytoplasmic free calcium concentration is a critical trigger for the initiation of such vital responses as muscle contraction and transcription. L-type Ca(v)1.2 calcium channels are complexes of the pore-forming α(1C) subunit associated with cytosolic Ca(v)β subunits. All major Ca(v)βs share a highly homologous membrane associated guanylate kinase-like (MAGUK) domain that binds to α(1C) at the α-interaction domain (AID), a short motif in the linker between transmembrane repeats I and II. In this study we show that Ca(v)β subunits form multimolecular homo- and heterooligomeric complexes in human vascular smooth muscle cells expressing native calcium channels and in Cos7 cells expressing recombinant Ca(v)1.2 channel subunits. Ca(v)βs oligomerize at the α(1C) subunits residing in the plasma membrane and bind to the AID. However, Ca(v)β oligomerization occurs independently on the association with α(1C). Molecular structures responsible for Ca(v)β oligomerization reside in 3 regions of the guanylate kinase subdomain of MAGUK. An augmentation of Ca(v)β homooligomerization significantly increases the calcium current density, while heterooligomerization may also change the voltage-dependence and inactivation kinetics of the channel. Thus, oligomerization of Ca(v)β subunits represents a novel and essential aspect of calcium channel regulation.

Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex

Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.

Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking

The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor.

Reciprocal Interactions Regulate Targeting of Calcium Channel β Subunits and Membrane Expression of α1 Subunits in Cultured Hippocampal Neurons

Auxiliary beta subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca(2+) channels in heterologous cells. In brain, all four beta isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of beta subunits and their role in membrane expression of Ca(V)1.2 alpha(1) subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous beta subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged beta subunits demonstrated that all beta subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca(V)1.2, thus indicating a great promiscuity in alpha(1)-beta interactions. In contrast, restricted axonal targeting of beta(1) and weak colocalization of beta(4b) with Ca(V)1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged Ca(V)1.2 was strongly enhanced by all beta subunits in an isoform-specific manner. Conversely, mutating the alpha-interaction domain of Ca(V)1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a beta subunit with the alpha-interaction domain is absolutely essential for membrane expression of alpha(1) subunits, as well as for the subcellular localization of beta subunits, which by themselves possess little or no targeting properties.

Identification and Functional Analysis of Type III Effector Proteins in Mesorhizobium loti

Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, possesses a cluster of genes (tts) that encode a type III secretion system (T3SS). In the presence of heterologous nodD from Rhizobium leguminosarum and a flavonoid naringenin, we observed elevated expression of the tts genes and secretion of several proteins into the culture medium. Inoculation experiments with wild-type and T3SS mutant strains revealed that the presence of the T3SS affected nodulation at a species level within the Lotus genus either positively (L. corniculatus subsp. frondosus and L. filicaulis) or negatively (L. halophilus and two other species). By inoculating L. halophilus with mutants of various type III effector candidate genes, we identified open reading frame mlr6361 as a major determinant of the nodulation restriction observed for L. halophilus. The predicted gene product of mlr6361 is a protein of 3,056 amino acids containing 15 repetitions of a sequence motif of 40 to 45 residues and a shikimate kinase-like domain at its carboxyl terminus. Homologues with similar repeat sequences are present in the hypersensitive-response and pathogenicity regions of several plant pathogens, including strains of Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas species. These results suggest that L. halophilus recognizes Mlr6361 as potentially pathogen derived and subsequently halts the infection process.

Ca2+/Calmodulin-dependent Protein Kinase II Binds to and Phosphorylates a Specific SAP97 Splice Variant to Disrupt Association with AKAP79/150 and Modulate α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor (AMPAR) Activity

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) promotes trafficking and activation of the GluR1 subunit of alpha-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) during synaptic plasticity. GluR1 is also modulated in parallel by multiprotein complexes coordinated by synapse-associated protein 97 (SAP97) that contain A-kinase anchoring protein 79/150 (AKAP79/150), protein kinase A, and protein phosphatase 2B. Here we show that SAP97 is present in CaMKII immune complexes isolated from rodent brain as well as from HEK293 cells co-expressing CaMKIIalpha and SAP97. CaMKIIalpha phosphorylated recombinant SAP97 within immune complexes in vitro and in intact cells. Four alternative mRNA splice variants of SAP97 expressing combinations of four inserts (I2, I3, I4, I5) in the U5 region between Src homology 3 (SH3) and guanylyl kinase-like (GK) domains were identified in rat brain at postnatal day 21. CaMKIIalpha preferentially phosphorylated a full-length SAP97 and a glutathione S-transferase (GST) fusion protein containing the I3 and I5 inserts (SAP97-I3I5 and GST-SH3-I3I5-GK, respectively) and also specifically interacted with GST-SH3-I3I5-GK compared with GST proteins containing other naturally occurring insert combinations. AKAP79/150 also directly and specifically bound only to GST-SH3-I3I5-GK, but CaMKII phosphorylation of GST-SH3-I3I5-GK prevented this interaction. AKAP79-dependent down-regulation of GluR1 AMPAR currents was ablated by overexpression of SAP97-I2I5 (which does not bind AKAP79) or by infusion of active CaMKIIalpha. Collectively, the data suggest that CaMKIIalpha targets a specific SAP97 splice variant to disengage AKAP79/150 from regulating GluR1 AMPARs, providing new insight into protein-protein interactions and phosphorylation events that are required for normal regulation of glutamatergic synaptic transmission, learning, and memory.

104. LRGUK - A NOVEL GENE INVOLVED IN MALE FERTILITY

Infertility affects a large number of Australian men. The causative factor in many of these cases is likely to be genetic in origin. As such, the identification and characterisation of novel genes involved in male fertility represents an important area of research. We have undertaken an N-ethyl-nitrosourea (ENU) mutagenesis screen to identify novel genes involved in male fertility. From this screen we have identified a mouse line that we have designated ‘Kaos’. Male mice that are homozygous for the Kaos mutation are infertile as a result of severely disrupted spermatogenesis, with Kaos homozygote males containing only 20% of the normal number of elongated spermatids in their testes, when examined by nuclear resistance to Triton X-100 solubilisation. In contrast, Kaos homozygote females are fertile. To identify the Kaos causal mutation, we used a combination of DNA linkage analysis and candidate gene sequencing. A point mutation was identified in exon 14 of the Leucine-rich Repeats and Guanylate Kinase domain containing (Lrguk) gene, which introduces a pre-mature stop codon into the Lrguk coding sequence. The function of the LRGUK protein is currently unknown. However preliminary expression and phenotype analysis suggests that Lrguk may have a role in spermiogenesis (i.e. haploid germ cell development). The deduced LRGUK protein contains several potentially important domains including a Guanylate Kinase-like (GK) domain and a number of leucine-rich repeats. The GK-like domain has some homology to guanylate kinase, a metabolic enzyme involved in purine nucleotide metabolism. Studies are currently underway to determine whether the LRGUK protein possesses guanylate kinase enzyme activity. In other proteins, the GK-like domain has evolved into mediating protein-protein interactions (1), and current studies are also aimed at examining this possibility. These studies should provide insight into the function of LRGUK and its role in spermatogenesis. (1) Montgomery J, et al. (2004). MAGUKs in synapse assembly and function: an emerging view. Cell Mol Life Sci. 61(7–8): 911–929.

Ligand-mediated endocytosis and intracellular sequestration of guanylyl cyclase/natriuretic peptide receptors: role of GDAY motif

The guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), also referred to as GC-A, is a single polypeptide molecule having a critical function in blood pressure regulation and cardiovascular homeostasis. GC-A/NPRA, which resides in the plasma membrane, consists of an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular cytoplasmic region containing a protein kinase-like homology domain (KHD) and a guanylyl cyclase (GC) catalytic domain. After binding with atrial and brain natriuretic peptides (ANP and BNP), GC-A/NPRA is internalized and sequestered into intracellular compartments. Therefore, GC-A/NPRA is a dynamic cellular macromolecule that traverses different subcellular compartments through its lifetime. This review describes the roles of short-signal sequences in the internalization, trafficking, and intracellular redistribution of GC-A/NPRA from cell surface to cell interior. Evidence indicates that, after internalization, the ligand-receptor complexes dissociate inside the cell and a population of GC-A/NPRA recycles back to the plasma membrane. Subsequently, the disassociated ligands are degraded in the lysosomes. However, a small percentage of the ligand escapes the lysosomal degradative pathway, and is released intact into culture medium. Using pharmacologic and molecular perturbants, emphasis has been placed on the cellular regulation and processing of ligand-bound GC-A/NPRA in terms of receptor trafficking and down-regulation in intact cells. The discussion is concluded by examining the functions of short-signal sequence motifs in the cellular life-cycle of GC-A/NPRA, including endocytosis, trafficking, metabolic processing, inactivation, and/or down-regulation in model cell systems.

Allosteric Control of Regulated Scaffolding in Membrane-Associated Guanylate Kinases

Membrane-associated guanylate kinases (MAGUKs) organize protein complexes at specific cellular sites by regulating interactions with their COOH-terminal guanylate kinase-like domains (GKs). Negative regulation of MAGUK GKs by an adjacent Src homology 3 domain (SH3) is critical for function, yet the mechanism is poorly understood. To gain insight into this process, we investigated SH3 regulation of the Discs large (Dlg) GK. Mutational analysis revealed that the binding site of the SH3-inhibited GK ligand GukHolder (GukH) is opposite the SH3 interacting surface, indicating that the SH3 does not directly occlude GukH binding. We screened for constitutively active SH3GK variants using yeast two-hybrid and a cell polarity/mitotic spindle orientation assay. Residues in both the SH3 and GK are required to maintain SH3GK inhibition, including those distant from both the SH3-GK and GK-GukH interaction sites. Activating mutations do not alter the ability of the SH3 and GK to interact in trans. On the basis of these observations, we propose that the SH3 modulates GK allostery to control its function.

Expression, purification, and characterization of the intra-cellular domain of the ANP receptor

The membrane-bound atrial natriuretic peptide receptor (GCA) catalyzes the formation of cGMP from GTP in response to natriuretic peptide hormones. Previous structural studies have focused on the extra-cellular hormone binding domain of this receptor whereas its intra-cellular domain has not yet been amenable to such studies. We report here the baculovirus expression and purification of the GCA intra-cellular domain construct GCA ID comprising the complete intra-cellular region which includes the kinase-homology domain, coiled-coil region, and catalytic cyclase domain. The intra-cellular domain was enzymatically characterized in terms of guanylyl cyclase activity and the effects of ATP, manganese, and Triton X-100. Our results indicate that the activity of the intra-cellular domain of the ANP receptor is about 2 fold less active compared to a truncated cyclase domain construct lacking the kinase-like domain that was also expressed and purified. In addition, unlike the full length receptor, the intra-cellular domain could not be activated by Triton X-100/Mn 2+ or its activity stimulated by ATP. These data therefore indicate that the major part of the transition from the basal state to the fully, ANP/ATP-dependent, activated state as well its stimulation/enhancement by Triton X-100/Mn 2+ requires the full length receptor. These receptor insights could aid in the development of novel therapeutics as the GCA receptor is a key drug target for cardiovascular diseases.

Analysis of the phytochrome gene family in Ceratodon purpureus by gene targeting reveals the primary phytochrome responsible for photo- and polarotropism

Using gene targeting by homologous recombination in Ceratodon purpureus, we were able to knock out four phytochrome photoreceptor genes independently and to analyze their function with respect to red light dependent phototropism, polarotropism, and chlorophyll content. The strongest phenotype was found in knock-out lines of a newly described phytochrome gene termed CpPHY4 lacking photo- and polarotropic responses at moderate fluence rates. Eliminating the atypical phytochrome gene CpPHY1, which is the only known phytochrome-like gene containing a putative C-terminal tyrosine kinase-like domain, affects red light-induced chlorophyll accumulation. This result was surprising, since no light dependent function was ever allocated to this unusual gene.

Comparative kinomics of human and chimpanzee reveal unique kinship and functional diversity generated by new domain combinations

BackgroundPhosphorylation by protein kinases is a common event in many cellular processes. Further, many kinases perform specialized roles and are regulated by non-kinase domains tethered to kinase domain. Perturbation in the regulation of kinases leads to malignancy. We have identified and analysed putative protein kinases encoded in the genome of chimpanzee which is a close evolutionary relative of human.ResultThe shared core biology between chimpanzee and human is characterized by many orthologous protein kinases which are involved in conserved pathways. Domain architectures specific to chimp/human kinases have been observed. Chimp kinases with unique domain architectures are characterized by deletion of one or more non-kinase domains in the human kinases. Interestingly, counterparts of some of the multi-domain human kinases in chimp are characterized by identical domain architectures but with kinase-like non-kinase domain. Remarkably, out of 587 chimpanzee kinases no human orthologue with greater than 95% sequence identity could be identified for 160 kinases. Variations in chimpanzee kinases compared to human kinases are brought about also by differences in functions of domains tethered to the catalytic kinase domain. For example, the heterodimer forming PB1 domain related to the fold of ubiquitin/Ras-binding domain is seen uniquely tethered to PKC-like chimpanzee kinase.ConclusionThough the chimpanzee and human are evolutionary very close, there are chimpanzee kinases with no close counterpart in the human suggesting differences in their functions. This analysis provides a direction for experimental analysis of human and chimpanzee protein kinases in order to enhance our understanding on their specific biological roles.