MEKK1 interacts with alpha-actinin and localizes to stress fibers and focal adhesions.

Mitogen-activated protein (MAP) kinases orchestrate the effects of many extracellular stimuli on cells. The serine/threonine protein kinase MEKK1 is an upstream activator of the MAP kinases c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), extracellular signal-regulated kinase (ERK), and p38 as well as NF-kappa B. In a yeast two-hybrid interaction screen to identify proteins that bind to an N-terminal fragment of MEKK1 (amino acids 1-719), the actin-crosslinking protein alpha-actinin was identified as a MEKK1-binding protein. Over-expressed MEKK1 co-immunoprecipitated with alpha-actinin in cell lysates. Both endogenous and over-expressed MEKK1 colocalized with alpha-actinin along actin stress fibers and at focal adhesions. Residues 221-559 of MEKK1 bound to purified alpha-actinin in vitro, indicating that the interaction is direct, and this fragment localized to actin filaments in cells. MEKK1 kinase activity was not required for association with actin filaments, because a catalytically inactive mutant of MEKK1 (MEKK1 D1369A) localized to stress fibers. These results provide strong evidence for the interaction between MEKK1 and alpha-actinin. Thus, restriction of the kinase to the actin cytoskeleton may serve to regulate its specificity towards downstream targets.

HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation.

NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.

Cloning of rat MEK kinase 1 cDNA reveals an endogenous membrane-associated 195-kDa protein with a large regulatory domain.

The coding sequence of rat MEK kinase 1 (MEKK1) has been determined from multiple, independent cDNA clones. The cDNA is full-length based on the presence of stop codons in all three reading frames of the 5' untranslated region. Probes from the 5' and the 3' coding sequences both hybridize to a 7-kb mRNA. The open reading frame is 4.5 kb and predicts a protein with molecular mass of 161,225 Da, which is twice the size of the previously published MEKK1 sequence and reveals 801 amino acids of novel coding sequence. The novel sequence contains two putative pH domains, two proline-rich regions, and a cysteine-rich region. Antisera to peptides derived from this new sequence recognize an endogenous protein in human and rodent cells of 195 kDa, consistent with the size of the expressed rat MEKK1 clone. Endogenous and recombinant rat MEKK1 are enriched in membranes; little of either is found in soluble fractions. Expression of recombinant rat MEKK1 leads to activation of three mitogen-activated protein kinase modules in the order c-Jun N-terminal kinase/stress-activated protein kinase > p38 mitogen-activated protein kinase = extracellular signal-regulated kinase 2.

orb is required for anteroposterior and dorsoventral patterning during Drosophila oogenesis.

We describe mutations in the orb gene, identified previously as an ovarian-specific member of a large family of RNA-binding proteins. Strong orb alleles arrest oogenesis prior to egg chamber formation, an early step of oogenesis, whereas females mutant for a maternal-effect lethal orb allele lay eggs with ventralized eggshell structures. Embryos that develop within these mutant eggs display posterior patterning defects and abnormal dorsoventral axis formation. Consistent with such embryonic phenotypes, orb is required for the asymmetric distribution of oskar and gurken mRNAs within the oocyte during the later stages of oogenesis. In addition, double heterozygous combinations of orb and grk or orb and top/DER alleles reveal that mutations in these genes interact genetically, suggesting that they participate in a common pathway. Orb protein, which is localized within the oocyte in wild-type females, is distributed ubiquitously in stage 8-10 orb mutant oocytes. These data will be discussed in the context of a model proposing that Orb is a component of the cellular machinery that delivers mRNA molecules to specific locations within the oocyte and that this function contributes to both D/V and A/P axis specification during oogenesis.