RICK, a novel protein kinase containing a caspase recruitment domain, interacts with CLARP and regulates CD95-mediated apoptosis

Signaling through the CD95/Fas/APO-1 death receptor plays a critical role in the homeostasis of the immune system. RICK, a novel protein kinase that regulates CD95-mediated apoptosis was identified and characterized. RICK is composed of an N-terminal serine-threonine kinase catalytic domain and a C-terminal region containing a caspase-recruitment domain. RICK physically interacts with CLARP, a caspase-like molecule known to bind to Fas-associated protein with death domain (FADD) and caspase-8. Expression of RICK promoted the activation of caspase-8 and potentiated apoptosis induced by Fas ligand, FADD, CLARP, and caspase-8. Deletion mutant analysis revealed that both the kinase domain and caspase-recruitment domain were required for RICK to promote apoptosis. Significantly, expression of a RICK mutant in which the lysine of the putative ATP-binding site at position 38 was replaced by a methionine functioned as an inhibitor of CD95-mediated apoptosis. Thus, RICK represents a novel kinase that may regulate apoptosis induced by the CD95/Fas receptor pathway.     

GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP

We have identified a mammalian protein called GIPC (for GAIP interacting protein, C terminus), which has a central PDZ domain and a C-terminal acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifically interacts with RGS-GAIP, a GTPase-activating protein (GAP) for Galphai subunits recently localized on clathrin-coated vesicles. Analysis of deletion mutants indicated that the PDZ domain of GIPC specifically interacts with the C terminus of GAIP (11 amino acids) in the yeast two-hybrid system and glutathione S-transferase (GST)-GIPC pull-down assays, but GIPC does not interact with other members of the RGS (regulators of G protein signaling) family tested. This finding is in keeping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of membrane fractions prepared from HeLa cells, we found that there are two pools of GIPC-a soluble or cytosolic pool (70%) and a membrane-associated pool (30%). By immunofluorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells reflecting, respectively, the existence of soluble and membrane-associated pools. By immunoelectron microscopy the membrane pool of GIPC is associated with clusters of vesicles located near the plasma membrane. These data provide direct evidence that the C terminus of a RGS protein is involved in interactions specific for a given RGS protein and implicates GAIP in regulation of additional functions besides its GAP activity. The location of GIPC together with its binding to GAIP suggest that GAIP and GIPC may be components of a G protein-coupled signaling complex involved in the regulation of vesicular trafficking. The presence of an ACP domain suggests a putative function for GIPC in the acylation of vesicle-bound proteins.     

P-CIP1, a novel protein that interacts with the cytosolic domain of peptidylglycine alpha-amidating monooxygenase, is associated with endosomes

The cytosolic domain of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) contains signals that direct its trafficking in the secretory and endosomal pathways. Using the yeast two-hybrid system, Alam et al. (Alam, M. R., Caldwell, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636) identified three proteins that interact with a fragment of the PAM cytosolic domain containing these targeting signals. We cloned the rat and human cDNAs encoding PAM COOH-terminal interactor protein-1 (P-CIP1). Both cDNAs contain an open reading frame that encodes a novel protein of 435 amino acids. The P-CIP1 protein is highly conserved from rat to human (85% identity) but does not display significant homology to proteins in the GenBank data base. In vitro, P-CIP1 interacts with the cytosolic domain of wild type PAM-1, but does not interact with mutant PAM-1 proteins that fail to target correctly when expressed in endocrine cells. P-CIP1 contains multiple consensus serine/threonine phosphorylation sites and a region predicted to form a coiled-coil at the COOH terminus. When expressed in endocrine cells or fibroblasts, P-CIP1 is distributed in a punctate pattern in the perinuclear area but does not significantly overlap the distribution of transfected wild type PAM-1. The distribution of P-CIP1 displays significant overlap with the distribution of the secretory carrier membrane proteins, internalized Texas Red-conjugated transferrin, and Rab11. The data suggest that P-CIP1 associates with vesicles in the recycling endosomal pathway, and may play a role in regulating the trafficking of integral membrane PAM.     

ALL-1 interacts with unr, a protein containing multiple cold shock domains

The ALL-1 gene is involved in human acute leukemia through chromosome translocations and fusion to partner genes, or through partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which transregulates the homeotic genes of the Antennapedia and bithorax complexes controlling body segment identity. ALL-1 encodes a very large protein of 3968 amino acids which presumably interacts with many proteins. Here we applied yeast two hybrid screening to identify proteins interacting with the N-terminal segment of ALL-1. One protein obtained in this way was the product of the unr gene. This protein consists of multiple repeats homologous to the cold shock domain (CSD), a motif common to some bacterial and eukaryotic nucleic acids-binding proteins. The minimal region on unr required for the interaction with ALL-1 included two CSD and two intervening polypeptides. The interaction was confirmed by in vitro binding studies, and by coimmunoprecipitation from COS cells overexpressing the relevant segments of the two proteins. These results suggest that unr is involved in an interaction of ALL-1 with DNA or RNA.     

Identification and purification of a spinach chloroplast DNA-binding protein that interacts specifically with the plastid psaA-psaB-rps14 promoter region

OBJECTIVE: To catalog a series of rare lesions of the posterior fossa that appeared with unusual initial retrocochlear symptoms and signs and to make the reader more aware of these unusual lesions with a view to improving initial assessment and treatment planning. STUDY DESIGN: The study was a retrospective case review of seven patients. SETTING: Multidisciplinary team evaluation in a tertiary hospital referral center. PATIENTS: Patients with unusual lesions of the cerebellopontine angle and posterior fossa with initial retrocochlear symptoms and signs were included. INTERVENTIONS: Diagnostic and therapeutic. MAIN OUTCOME MEASURES: Hearing preservation and balance function. RESULTS: The rare lesions presented include two aneurysms of the anterior inferior cerebellar artery, one giant basilar artery aneurysm, and one each of the following neoplasms: endodermal cyst, choroid plexus papilloma, cavernous angioma, and ependymoma. CONCLUSIONS: A close working relationship among the otolaryngologist, neurotologist, neurosurgeon, and neuroradiologist is necessary to accurately evaluate these unusual cerebellopontine angle lesions and effect the best treatment outcome.     

Lambda repressor N-terminal DNA-binding domain as an assay for protein transmembrane segment interactions in vivo

OBJECTIVE: To critically review the English-language literature and describe the current diagnosis, prevalence, etiology, and treatment of antisperm antibodies (ASA). DESIGN: A comprehensive literature search of the English-language literature published between 1966 and December 1997 was performed on MEDLINE. Articles were also located via bibliographies of published works. RESULT(S): Data were excerpted from articles identified by MEDLINE search. The diagnosis, prevalence, etiology, and treatment of ASA are described. CONCLUSION(S): There is sufficient evidence that ASA impair fertility in couples with unexplained infertility. A number of different methodologies are available, which may be used in their detection. However, in many cases, test interpretation is subjective. Although there is not enough evidence to support systemic treatment for ASA, application of a variety of assisted reproductive technologies improves outcome.     

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.     

The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein

Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins [1-3]. Connexin43 (Cx43) is the most widely expressed and the most well-studied member of this family. Cx43-based cell-cell communication is regulated by growth factors and oncogenes [3-5], although the underlying mechanisms are poorly understood as cellular proteins that interact with connexins have yet to be identified. The carboxy-terminal cytosolic domain of Cx43 contains several phosphorylation sites and potential signalling motifs. We have used a yeast two-hybrid protein interaction screen to identify proteins that bind to the carboxy-terminal tail of Cx43 and thereby isolated the zona occludens-1 (ZO-1) protein. ZO-1 is a 220 kDa peripheral membrane protein containing multiple protein interaction domains including three PDZ domains and a Src homology 3 (SH3) domain [6-9]. The interaction of Cx43 with ZO-1 occurred through the extreme carboxyl terminus of Cx43 and the second PDZ domain of ZO-1. Cx43 associated with ZO-1 in Cx43-transfected COS7 cells, as well as endogenously in normal Rat-1 fibroblasts and mink lung epithelial cells. Confocal microscopy revealed that endogenous Cx43 and ZO-1 colocalised at gap junctions. We suggest that ZO-1 serves to recruit signalling proteins into Cx43-based gap junctions.     

The lissencephaly gene product Lis1, a protein involved in neuronal migration, interacts with a nuclear movement protein, NudC

Important clues to how the mammalian cerebral cortex develops are provided by the analysis of genetic diseases that cause cortical malformations [1-5]. People with Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) have a hemizygous deletion or mutation in the LIS1 gene [3,6]; both conditions are characterized by a smooth cerebral surface, a thickened cortex with four abnormal layers, and misplaced neurons [7,8]. LIS1 is highly expressed in the ventricular zone and the cortical plate [9,10], and its product, Lis1, has seven WD repeats [3]; several proteins with such repeats have been shown to interact with other polypeptides, giving rise to multiprotein complexes [11]. Lis1 copurifies with platelet-activating factor acetylhydrolase subunits alpha 1 and alpha 2 [12], and with tubulin; it also reduces microtubule catastrophe events in vitro [13]. We used a yeast two-hybrid screen to isolate new Lis1-interacting proteins and found a mammalian ortholog of NudC, a protein required for nuclear movement in Aspergillus nidulans [14]. The specificity of the mammalian NudC-Lis1 interaction was demonstrated by protein-protein interaction assays in vitro and by co-immunoprecipitation from mouse brain extracts. In addition, the murine mNudC and mLis1 genes are coexpressed in the ventricular zone of the forebrain and in the cortical plate. The interaction of Lis1 with NudC, in conjunction with the MDS and ILS phenotypes, raises the possibility that nuclear movement in the ventricular zone is tied to the specification of neuronal fates and thus to cortical architecture.