BIOLOGICAL OVERVIEW

spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire contains two domains with similarity to the actin monomer-binding WH2 domain, and Spire binds to actin in the interaction trap system and in vitro. In addition, Spire interacts with the rho family GTPases RhoA, Rac1 and Cdc42 in the interaction trap system. This evidence supports the model that Spire links rho family signaling to the actin cytoskeleton (Wellington, 1999).

Previous work has shown that in spire mutants, the microtubules bundle prematurely, during stage 8 at the cortex. This premature microtubule bundling is accompanied by rapid, microtubule-dependent swirling of the cytoplasm (Theurkauf, 1994). Both the bundling of microtubules and cytoplasmic streaming are normally seen later in stage 10 wild-type oocytes (Wellington, 1999).

A bi-directional signaling process occurs between the oocyte and the posterior follicle cells to establish the posterior pole of the egg. Phenotypes indicative of a defect in this signaling process include transformation of the posterior follicle cells into an anterior follicle cell fate, misorganization of the microtubules at stage 6, localization of Oskar mRNA to the center of the oocyte, localization of Bicoid mRNA to the posterior pole, and premature cytoplasmic streaming. The posterior follicle cell fates are established correctly in spire. In contrast to a previous report (Kim-Ha, 1991), a central spot of Oskar mRNA staining was not observed. Bicoid mRNA localization appears relatively normal in spire. These results suggest that signaling between the posterior follicle cells and the oocyte is not abnormal in spire mutants. In spire mutant oocytes microtubules sometimes show abnormal distributions during stage 6, but it is thought that this probably reflects an earlier manifestation of the known spire microtubule defect (Wellington, 1999).

WH2 domains, like those found in Spire, have been found in the Wiskott-Aldrich syndrome protein (WASP), verprolin, Scar-1 (see Drosophila SCAR), and a number of other proteins of unknown function. The WH2 domains of N-WASP and of Scar-1 have been shown to bind directly to G-actin in vitro. In addition, Spir binds to unpolymerized actin in vitro. Although Spir is capable of binding to actin monomers through its WH2 domain, no defects in the actin cytoskeleton have been observed in spir mutants, suggesting a number of possibilities. The defects may be in actin structures that are difficult to observe, such as the cell cortex. In fact, spir phenotypes can be mimicked by treatment with cytochalasin D, a drug that affects the polymerization state of actin; defects in the actin cytoskeleton in cytochalasin D-treated oocytes have not been observed. Alternatively, spir may act downstream of the actin cytoskeleton and, thus, not change it. Finally, in vitro experiments have shown that, in the absence of the neighboring cofilin homology and acidic domains, the two WH2 domains of N-WASP have either no effect on or slowly depolymerize filamentous actin. Since Spir is lacking the cofilin homology and acidic domains, Spir may have no effect on filamentous actin or only a minor one (Wellington, 1999 and references therein).

It is becoming more apparent that a relationship exists between the actin cytoskeleton and premature microtubule-dependent cytoplasmic streaming. The premature cytoplasmic streaming phenotype of spir can be mimicked by the addition of cytochalasin D, a drug that depolymerizes actin filaments. Additional evidence that the actin cytoskeleton is involved in repressing microtubule bundling and streaming comes from analysis of mutant phenotypes of genes linked to the actin cytoskeleton. In addition to spir, mutants in chickadee that encode profilin and capu, which is thought to bind to profilin, also exhibit these microtubule behaviors. While phenotypes for cdc42 and rac1 have been described during oogenesis, their effects on patterning during oogenesis are unknown. The finding that Spir interacts with rho family GTPases suggests that at least one of the rho family GTPases is functioning in patterning. Further genetic and biochemical studies will be required to determine the nature of Spir's interaction with rho family GTPases in vivo (Wellington, 1999).

A direct link between JNK signaling and actin organization has not been found previously. The Jun N-terminal kinase (JNK, known as Basket in Drosophila) is a downstream effector of Rac and Cdc42 GTPases involved in actin reorganization. Basket plays a role in the regulation of cell shape changes and actin reorganization during the process of dorsal closure. One potential mechanism for induction of cytoskeletal changes by Basket is via transcriptional activation of the decapentaplegic gene, a member of the TGFbeta superfamily. Spire, termed p150-Spire in this study, has a docking site for Basket/JNK, at its carboxy-terminal end. In mouse fibroblasts, p150-Spir colocalizes with F-actin; its overexpression induces clustering of filamentous actin around the nucleus. p150Spir (Otto, 2000) is slightly longer than the long form characterized by Wellington (1999). When coexpressed with p150-Spire in NIH 3T3 cells, JNK translocates to and colocalizes with p150-Spire at discrete spots around the nucleus. Carboxy-termina l sequences of p150-Spir are phosphorylated by JNK both in vitro and in vivo. It is concluded that p150-Spir is a downstream target of JNK function and provides a direct link between JNK and actin organization (Otto, 2000).

p150Spire was identified in a yeast two-hybrid screen to search for novel Basket interaction partners. The cDNA encoding p150Spire contains an open reading frame of 1,020 amino acids. Transient transfection experiments in mouse fibroblasts reveal that the cDNA directs expression of a protein that migrates with an apparent molecular weight of approximately 150 kDa on an SDS-polyacrylamide gel. The spire gene exhibits significant homology to a maternal gene pem-5 isolated from the ascidian Ciona savignyi (phylum Urochordata). Comparison of Drosophila p150Spire sequences to human EST clones reveals a high conservation between the p150Spire sequences of Drosophila and human (Otto, 2000).

The p150Spire protein contains an acidic domain, a cluster of four WH2 domains, a modified FYVE zinc finger domain and a carboxy-terminal DEJL motif (docking site for Erk and JNK containing an LXL motif). WH2 domains bind monomeric actin: WH2 family proteins such as WASP and WAVE are involved in actin reorganization. Sequences encompassing the Spir WH2 domains interact directly with monomeric actin. As has been shown for WASP and WAVE, transient expression of p150Spire in adherent mammalian cells induces clusters of filamentous actin around the nucleus that colocalize with p150Spire in all transfected cells. A modified FYVE zinc-finger motif is located in the central region of the protein. The modified structure lacks a pocket of basic amino acids between cysteines 3 and 4 of the FYVE finger structures that is necessary for the binding of phosphatidylinositol 3-phosphate (Otto, 2000).

In this study, p150Spire was identified in a yeast two-hybrid screen as a DJNK-interacting protein. The smallest fragment that still binds DJNK consists of the carboxy-terminal 53 amino acids. A DEJL motif, characterized by a cluster of basic amino acids amino-terminal to an L/I-X-L/I motif (in the single-letter amino acid code), is located in this carboxy-terminal sequence (amino acids 997-1014 of p150Spire, KQKRSSARNRTIQNLTLD). DEJL domains have been shown to mediate the docking of the Erk and JNK mitogen-activated protein (MAP) kinases to either activating kinases or substrate proteins. To further characterize the interaction between p150Spire and JNK, the localization of the two proteins was determined when transiently expressed in NIH 3T3 cells. p150Spire accumulates in punctate spots located around the nucleus. Expression of a fusion protein between glutathione-S-transferase and JNK (GST-JNK) reveals both a cytoplasmic and nuclear distribution of the kinase. In the presence of p150Spire, GST-JNK is translocated from these areas and accumulates at places where p150Spire is located. The colocalization of the proteins is detected in all cells expressing both proteins but is not found in cells coexpressing GST alone with p150Spire (Otto, 2000).

Erk and JNK MAP kinases are recruited to substrate proteins via docking sites, enabling the kinases to phosphorylate serine or threonine residues adjacent to prolines (S/TP motifs). As p150Spire contains a JNK docking site (the DEJL motif) and several potential S/TP phosphorylation motifs, attempts were made to determine whether p150Spire is a phosphorylation target of JNK. A highly specific, constitutively active Erk can be generated by the fusion of Erk2 to its upstream activator Mek1 (Erk2-Mek1-LA). Similarly, JNK2 (rat) has been fused to its upstream activator MKK7 (mouse) via a linker region. The fusion protein, JNK-MKK7, is a constitutively active Jun N-terminal kinase. JNK-MKK7 phosphorylates amino-terminal c-Jun sequences in vitro and induces an electrophoretic mobility shift of the c-Jun protein when coexpressed in NIH 3T3 cells, indicating an in vivo c-Jun phosphorylation. MKK7 activates JNK by phosphorylating a TPY motif in the central region of JNK. JNK-MKK7 exhibits autophosphorylation and interacts with a phospho-specific antibody that recognizes activated JNK protein. A similar construct (JNKK2-JNK1) has been shown to be a specific, constitutively active c-Jun kinase (Otto, 2000).

An investigation was carried out to see whether p150Spire sequences are phosphorylated by JNK in vitro. Indeed, arsenite-activated JNK1, immunoprecipitated from NIH 3T3 cell lysates, phosphorylates a carboxy-terminal fragment of the p150Spire protein in an in vitro immune-complex kinase assay. Erk and p38 MAP kinases precipitated from the same lysates exhibit a basal phosphorylation activity that could not be increased by arsenite stimulation of the cells. Coexpression of p150Spire with MLK3-activated JNK2 [mixed lineage kinase (MLK3) is an upstream activator of JNKs] or JNK-MKK7 in mouse fibroblasts, induces several slower migrating forms of the p150Spire protein during SDS-PAGE (Otto, 2000).

The electrophoretic mobility shift of p150Spire strongly suggests that it is phosphorylated by JNK on multiple sites. The slower migrating forms are abolished by phosphatase treatment, demonstrating that the mobility shifts are due to phosphorylation of p150Spire protein. Supporting these data, a kinase-inactive mutant of JNK-MKK7 (containing the JNK mutations K55A and K56A and the MKK7 mutation K76E, called JNK-MKK7-KD) does not induce an electrophoretic mobility shift of p150Spire. To analyse the specificity of JNK phosphorylation of p150Spire in vivo, p150Spire was coexpressed with the constitutively active Erk2-Mek1-LA fusion protein. Although expressed to the same levels as JNK-MKK7, Erk2-Mek1-LA induces only a very weak electrophoretic mobility shift of p150Spire, indicating a preferential phosphorylation of p150Spire by JNK (Otto, 2000).

In conclusion, the JNKs are downstream effectors of the Rac and Cdc42 GTPases involved in actin reorganization. Microinjection of activated Rac and Cdc42 proteins into cells induces changes in actin structures within minutes, before de novo protein synthesis. A WH2 family protein p150Spire, involved in actin reorganization, has been identified as a phosphorylation target of JNK activity. Strikingly, amino-terminal sequences of Spir interact with Rho family GTPases in yeast two-hybrid experiments (Wellington, 1999). One possible mechanism of actin regulation by Rho GTPases could therefore be the activation of a MAP kinase, which subsequently phosphorylates an actin regulator such as WAVE or p150Spire, thus regulating a role in either actin nucleation or the localization of actin regulating proteins. Future experiments will illuminate the cellular functions of p150Spire and its regulation by JNK phosphorylation (Otto, 2000).

GENE STRUCTURE

cDNA clone length - 2.7 and 4.5 kb

Bases in 5' UTR - 585 (long form)

Bases in 3' UTR - 1235 (long form)

PROTEIN STRUCTURE

Amino Acids - 585 and 990 (short and long form respectively; Wellington, 1999) and 1020 (p150-Spir; Otto, 2000)

Structural Domains

spir encodes two different proteins of 585 amino acids (aa) and 990 aa. Predicted coiled-coil regions are found at aa 281-294 and aa 932-947, and putative nuclear localization signals at aa 423, 424 and 579. Database searches reveal that spire is related to pem-5, a gene of unknown function in the ascidian Ciona savignyi (Satou, 1997). The proteins contain three regions of high similarity, and have been named SPEM1-3 (SPIRE and PEM). Pem-5 mRNA is localized to the posterior pole of the sea squirt. Since spir has a posterior group phenotype, it is of particular interest to know whether Spire mRNA localizes to the posterior pole of the oocyte. In situ hybridization using probes specific for the 4.5 kb and 2.7 kb mRNAs indicates that both Spire mRNAs are present in the follicle cells and nurse cells from region 2 of the germarium, but are not localized to the posterior pole of the oocyte. Spire is also related to human ESTs found in a variety of tissue types (Wellington, 1999).

Both the Simple Modular Architecture Research Tool (SMART) and BLAST searches reveal two regions in Spire that have a low level of sequence similarity to the WH2 or VPH regions of bovine N-WASP, a protein related to the Wiskott Aldrich Syndrome protein. The WH2 region of N-WASP and the WASP-related protein Scar1 have been shown to bind actin monomers in vitro (Wellington, 1999 and references therein).

A novel DJNK-interacting protein, p150Spire, belongs to the Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2) family of proteins involved in actin reorganization. It is a multidomain protein with a cluster of four WH2 domains, a modified FYVE zinc-finger motif, and a DEJL motif that serves as a docking site for JNK, at its carboxy-terminal end (Otto, 2000).

EVOLUTIONARY HOMOLOGS

The posterior-vegetal cytoplasm of an ascidian egg contains maternal factors required for pattern formation and cell specification of the embryo. The isolation and characterization is reported of cDNA clones for novel maternal genes, posterior end mark 2 (pem-2), pem-4, pem-5 (a homolog of spire), and pem-6. These clones were obtained from a cDNA library of Ciona savignyi fertilized egg mRNAs subtracted with gastrula mRNAs by examining the localization of the corresponding mRNAs of randomly selected clonesusing whole-mount in situ hybridization. As in the case of pem, all of these mRNAs were localized in the posterior-vegetal cytoplasm of the egg, and they later marked the posterior end of early embryos. The predicted amino acid sequence suggests that PEM-2 contains a signal for nuclear localization, a src homology 3 (SH3) domain, and a consensus sequence of the CDC24 family guanine nucleotide dissociation stimulators (GDSs). PEM-4 has a signal for nuclear localization and three C2H2-type zinc finger motifs, while PEM-5 and PEM-6 show no similarity to known proteins. These results provide further evidence that the ascidian egg contains maternal messages that are localized in the posterior-vegetal cytoplasm (Satou, 1997).

The Wiskott-Aldrich homology domain 2 (WH2) family protein Spir and the formin Cappuccino belong to two distinct classes of actin organizers. Despite their functional classification as actin organizers, a major defect of Drosophila spire and cappuccino mutant oocytes is a failure in the orientation of microtubule plus ends towards the posterior pole. Mammalian homologues of spire are the spir-1 and spir-2 genes. The mouse and human formin-1 and formin-2 genes have high similarity to the cappuccino gene. The mouse formin-2 gene has been found to be expressed in the developing nervous system and in neuronal cells of the adult brain. By analyzing the expression of the spir-1 gene it has been shown that spir-1 and formin-2 have a nearly identical expression pattern during mouse embryogenesis and in the adult brain. In mouse embryos both genes are expressed in the developing nervous system. In the adult brain high expression of the genes was found in the Purkinje cells of the cerebellum and in neuronal cells of the hippocampus and dentate gyrus (Schumacker, 2004).

date revised: 2 June 2000

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