The genomic structure of the human SPEC1 gene reveals complex splicing and close promoter proximity to the AF1q translocation gene.

SPECs are small Cdc42 signaling molecules. In mammals, two genes, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acid residues, respectively. Here we report the expression and genomic organization of the human SPEC1 gene. Using Northern blot analysis, three major SPEC1 mRNA transcripts of 1.6, 3.3, and 6.3 kb were detected. Identification and sequencing of different sized SPEC1 cDNA clones revealed that the transcript size heterogeneity was due to alternative splicing in the 3'-untranslated region. In addition, a distinct SPEC1 splice variant from within the coding sequence, SPEC1-beta, was identified and detected in a variety of human tissues. Analysis of the genomic organization of SPEC1 revealed that the coding sequence of the SPEC1 isoform was derived from exons 2, 3 and 4, while the SPEC1-beta isoform was derived from exon 2 and a read-through event of intron 2. Examination of the 5'-end of the SPEC1 genomic sequence revealed that AF1q, a previously identified gene involved in translocations with the MLL (mixed-lineage leukemia) gene, was 631 bp away in a head-to-head orientation. This intergenic sequence containing the putative promoter region for both SPEC1 and AF1q genes did not contain a TATA box or CAAT box. Transfection experiments using an AF1q promoter luciferase reporter construct in a variety of cells including Cos1 cells, Jurkat T-cells, MCF-7 breast cancer cells, and NIH-3T3 fibroblasts showed no promoter activity. In contrast, a SPEC1 promoter luciferase reporter construct showed high levels of reporter activity in Cos1 and MCF-7 cells, low activity in NIH-3T3 fibroblasts and no activity in Jurkat T-cells. These promoter analyses suggest that although SPEC1 and AF1q genes share the same promoter region, they are not coordinately regulated.

Evolutionary expansion of CRIB-containing Cdc42 effector proteins.

Cdc42, a small GTPase, regulates actin polymerization and other signaling pathways through interaction with many different downstream effector proteins. Most of these effector proteins contain a Cdc42-binding domain, called a CRIB domain. Here, we describe the evolutionary analysis of these CRIB-containing proteins in yeast, worms, flies and humans. The number of CRIB-containing effector proteins increases from yeast to humans, involving both an increase within families and the emergence of new families. These evolutionary changes correlate with the development of the more complex signaling pathways present in higher organisms.

A new family of Cdc42 effector proteins, CEPs, function in fibroblast and epithelial cell shape changes.

Cdc42, a Rho GTPase, regulates the organization of the actin cytoskeleton by its interaction with several distinct families of downstream effector proteins. Here, we report the identification of four new Cdc42-binding proteins that, along with MSE55, constitute a new family of effector proteins. These molecules, designated CEPs, contain three regions of homology, including a Cdc42 binding domain and two unique domains called CI and CII. Experimentally, we have verified that CEP2 and CEP5 bind Cdc42. Expression of CEP2, CEP3, CEP4, and CEP5 in NIH-3T3 fibroblasts induced pseudopodia formation. Fibroblasts coexpressing dominant negative Cdc42 with CEP2 or expressing a Cdc42/Rac interactive binding domain mutant of CEP2 did not induce pseudopodia formation. In primary keratinocytes, CEP2- and CEP5-expressing cells showed reduced F-actin localization at the adherens junctions with an increase in thin stress fibers that extended the length of the cell body. Keratinocytes expressing CEPs also showed an altered vinculin distribution and a loss of E-cadherin from adherens junctions. Similar effects were observed in keratinocytes expressing constitutively active Cdc42, but were not seen with a Cdc42/Rac interactive binding domain mutant of CEP2. These results suggest that CEPs act downstream of Cdc42 to induce actin filament assembly leading to cell shape changes.

SPECs, small binding proteins for Cdc42.

The Rho GTPase, Cdc42, regulates a wide variety of cellular activities including actin polymerization, focal complex assembly, and kinase signaling. We have identified a new family of very small Cdc42-binding proteins, designated SPECs (for Small Protein Effector of Cdc42), that modulates these regulatory activities. The two human members, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acids, respectively. Both contain a conserved N-terminal region and a centrally located CRIB (Cdc42/Rac Interactive Binding) domain. Using a yeast two-hybrid system, we found that both SPECs interact strongly with Cdc42, weakly with Rac1, and not at all with RhoA. Transfection analysis revealed that SPEC1 inhibited Cdc42-induced c-Jun N-terminal kinase (JNK) activation in COS1 cells in a manner that required an intact CRIB domain. Immunofluorescence experiments in NIH-3T3 fibroblasts demonstrated that both SPEC1 and SPEC2 showed a cortical localization and induced the formation of cell surface membrane blebs, which was not dependent on Cdc42 activity. Cotransfection experiments demonstrated that SPEC1 altered Cdc42-induced cell shape changes both in COS1 cells and in NIH-3T3 fibroblasts and that this alteration required an intact CRIB domain. These results suggest that SPECs act as novel scaffold molecules to coordinate and/or mediate Cdc42 signaling activities.

Cloning, central nervous system expression and chromosomal mapping of the mouse PAK-1 and PAK-3 genes.

Two cDNAs encoding PAK kinases were isolated from a mouse embryo library by screening with a PCR-generated probe derived from the kinase domain of a rat PAK kinase. These cDNAs, designated PAK-1 and PAK-3, encode mouse PAK kinases of 545 and 544 amino acids, respectively. Both proteins possess an N-terminal Cdc42/Rac interacting binding domain (CRIB) and a C-terminal serine/threonine kinase domain. Comparison of the two mouse PAK kinases revealed that the proteins show 87% amino acid identity. Northern analysis of a multiple mouse tissue blot with a PAK-1 probe detected a 3.0kb transcript that was almost exclusively expressed in the brain and spinal cord compared to other tissues such as lung, liver and kidney. A similar pattern of central nervous system tissue expression of PAK-3 transcripts of 3.6 and 8kb was also observed. Analysis of two multilocus genetic crosses localized Pak1 and Pak3 to a position on chromosome 7 and X, respectively. The high level of PAK-1 and PAK-3 kinase expression in the mouse brain and spinal cord suggests a potentially important role for these kinases in the control of the cellular architecture and/or signaling in the central nervous system.