The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes.

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains.

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.

The regulatory subunit of protein kinase CK2 is a specific A-Raf activator.

Two protein kinases that are involved in proliferation and oncogenesis but so far were thought to be functionally independent are Raf and CK2. The Raf signaling pathway is known to play a critical role in such fundamental biological processes as cellular proliferation and differentiation. Abnormal activation of this pathway is potentially oncogenic. Protein kinase CK2 exhibits enhanced levels in solid human tumors and proliferating tissue. In a two-hybrid screen of a mouse-embryo cDNA library we detected an interaction between A-Raf and CK2beta subunit. This binding was specific, as no interaction between CK2beta and B-Raf or c-Raf-1 was observed. Regions critical for this interaction were localized between residues 550 and 569 in the A-Raf kinase domain. A-Raf kinase activity was enhanced 10-fold upon coexpression with CK2beta in Sf9 cells. The alpha subunit of CK2 abolishes this effect. This is the first demonstration of both a direct Raf-isoform-specific activation and a regulatory role for CK2beta independent of the CK2alpha subunit. The present data thus link two different protein kinases that were thought to work separately in the cell.

Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.

Differential regulation of Raf isozymes by growth versus differentiation inducing factors in PC12 pheochromocytoma cells.

PC12 pheochromocytoma cells possess four known MEK activators: A-, B-, c-Raf-1 and MEKK. In order to examine whether differentiation factors or growth factors have a Raf isozyme preference for activation of the mitogenic cytoplasmic Raf-MEK-MAPK protein kinase cascade, the activation kinetics of these enzymes in response to epidermal growth factor (EGF) and nerve growth factor (NGF) were compared. An initial activation of all three Raf kinases was noticed, but only A- and B-Raf showed sustained activation by NGF, which was not seen after EGF treatment. Furthermore, expression of oncogenic versions of all three Raf kinases as well, as a potentially Raf-independent MEK activator, v-Mos, leads to activation of MAPK and to differentiation of PC12 cells. These data suggest a differential regulation of Raf kinases and that probably no alternative Raf substrates are involved in differentiation processes of PC12 cells.

3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity.