Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in E. coli.

Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion: a new tumor entity.

We present clinicopathologic data on 10 pulmonary myxoid sarcomas, which are defined by distinctive histomorphologic features and characterized by a recurrent fusion gene, that appear to represent a distinct tumor entity at this site. The patients [7 female, 3 male; aged 27 to 67 y (mean, 45 y)] presented with local or systemic symptoms (n=5), symptoms from cerebral metastasis (1), or incidentally (2). Follow-up of 6 patients showed that 1 with brain metastasis died shortly after primary tumor resection, 1 developed a renal metastasis but is alive and well, and 4 are disease free after 1 to 15 years. All tumors involved pulmonary parenchyma, with a predominant endobronchial component in 8 and ranged from 1.5 to 4 cm. Microscopically, they were lobulated and composed of cords of polygonal, spindle, or stellate cells within myxoid stroma, morphologically reminiscent of extraskeletal myxoid chondrosarcoma. Four cases showed no or minimal atypia, 6 showed focal pleomorphism, and 5 had necrosis. Mitotic indices varied, with most tumors not exceeding 5/10 high-power fields. Tumors were immunoreactive for only vimentin and weakly focal for epithelial membrane antigen. Of 9 tumors, 7 were shown to harbor a specific EWSR1-CREB1 fusion by reverse transcription-polymerase chain reaction and direct sequencing, with 7 of 10 showing EWSR1 rearrangement by fluorescence in situ hybridization. This gene fusion has been described previously in 2 histologically and behaviorally different sarcomas: clear cell sarcoma-like tumors of the gastrointestinal tract and angiomatoid fibrous histiocytomas; however, this is a novel finding in tumors with the morphology we describe and that occur in the pulmonary region.