O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1

The human epigenetic cell-cycle regulator HCF-1 undergoes an unusual proteolytic maturation process resulting in stably associated HCF-1(N) and HCF-1(C) subunits that regulate different aspects of the cell cycle. Proteolysis occurs at six centrally located HCF-1(PRO)-repeat sequences and is important for activation of HCF-1(C)-subunit functions in M phase progression. We show here that the HCF-1(PRO) repeat is recognized by O-linked β-N-acetylglucosamine transferase (OGT), which both O-GlcNAcylates the HCF-1(N) subunit and directly cleaves the HCF-1(PRO) repeat. Replacement of the HCF-1(PRO) repeats by a heterologous proteolytic cleavage signal promotes HCF-1 proteolysis but fails to activate HCF-1(C)-subunit M phase functions. These results reveal an unexpected role of OGT in HCF-1 proteolytic maturation and an unforeseen nexus between OGT-directed O-GlcNAcylation and proteolytic maturation in HCF-1 cell-cycle regulation.     

Intramembrane cleavage of ephrinB3 by the human rhomboid family protease, RHBDL2

Rhomboid-1 is a serine protease that cleaves the membrane domain of the Drosophila EGF-family protein, Spitz, to release a soluble growth factor. Several vertebrate rhomboid-like proteins have been identified, although their substrates and functions remain unknown. The human rhomboid, RHBDL2, cleaves the membrane domain of Drosophila Spitz when the proteins are co-expressed in mammalian cells. However, the membrane domains of several mammalian EGF-family proteins were not cleaved by RHBDL2, suggesting that the endogenous targets of the human protease are not EGF-related factors. We demonstrate that the amino acid sequence at the luminal face of the membrane domain of a substrate protein determines whether it is cleaved by RHBDL2. Based on this finding, we predicted B-type ephrins as potential RHBDL2 substrates. We found that one of these, ephrinB3, was cleaved so efficiently by the protease that little ephrinB3 was detected on the surface of cells co-expressing RHBDL2. These results raise the possibility that RHBDL2-mediated proteolytic processing may regulate intercellular interactions between ephrinB3 and eph receptors.     

An Arabidopsis Rhomboid homolog is an intramembrane protease in plants

Regulated intramembrane proteolysis (RIP) is a fundamental mechanism for controlling a wide range of cellular functions. The Drosophila protein Rhomboid-1 (Rho-1) is an intramembrane serine protease that cleaves epidermal growth factor receptor (EGFR) ligands to release active growth factors. Despite differences in the primary structure of Rhomboid proteins, the proteolytic activity and substrate specificity of these enzymes has been conserved in diverse organisms. Here, we show that an Arabidopsis Rhomboid protein AtRBL2 has proteolytic activity and substrate specificity. AtRBL2 cleaved the Drosophila ligands Spitz and Keren, but not similar proteins like TGFalpha, when expressed in mammalian cells, leading to the release of soluble ligands into the medium. These studies provide the first evidence that the determinants of RIP are present in plants.     

MLL3, a new human member of the TRX/MLL gene family, maps to 7q36, a chromosome region frequently deleted in myeloid leukaemia

We characterized MLL3, a new human member of the TRX/MLL gene family. MLL3 is expressed in peripheral blood, placenta, pancreas, testes, and foetal thymus and is weakly expressed in heart, brain, lung, liver, and kidney. It encodes a predicted protein of 4911 amino acids containing two plant homeo domains (PHD), an ATPase alpha_beta signature, a high mobility group, a SET (Suppressor of variegation, Enhancer of zeste, Trithorax) and two FY (phenylalanine tyrosine)-rich domains. The amino acid sequence of the SET domain was used to obtain a phylogenetic tree of human MLL genes and their homologues in different species. MLL3 is closely related to human MLL2, Fugu mll2, a Caenorhabditis elegans predicted protein, and Drosophila trithorax-related protein. Interestingly, PHD and SET domains are frequently found in proteins encoded by genes that are rearranged in different haematological malignancies and MLL3 maps to 7q36, a chromosome region that is frequently deleted in myeloid disorders. Partial duplications of the MLL3 gene are found in the juxtacentromeric region of chromosomes 1, 2, 13, and 21.     

HCF-1 amino- and carboxy-terminal subunit association through two separate sets of interaction modules: involvement of fibronectin type 3 repeats

When herpes simplex virus infects permissive cells, the viral regulatory protein VP16 forms a specific complex with HCF-1, a preexisting nuclear protein involved in cell proliferation. The majority of HCF-1 in the cell is a complex of associated amino (HCF-1(N))- and carboxy (HCF-1(C))-terminal subunits that result from an unusual proteolytic processing of a large precursor polypeptide. Here, we have characterized the structure and function of sequences required for HCF-1(N) and HCF-1(C) subunit association. HCF-1 contains two matched pairs of self-association sequences called SAS1 and SAS2. One of these matched association sequences, SAS1, consists of a short 43-amino-acid region of the HCF-1(N) subunit, which associates with a carboxy-terminal region of the HCF-1(C) subunit that is composed of a tandem pair of fibronectin type 3 repeats, a structural motif known to promote protein-protein interactions. Unexpectedly, the related protein HCF-2, which is not proteolyzed, also contains a functional SAS1 association element, suggesting that this element does not function solely to maintain HCF-1(N) and HCF-1(C) subunit association. HCF-1(N) subunits do not possess a nuclear localization signal. We show that, owing to a carboxy-terminal HCF-1 nuclear localization signal, HCF-1(C) subunits can recruit HCF-1(N) subunits to the nucleus.     

The COMPASS family of H3K4 methylases in Drosophila

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila melanogaster Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). Here, we report the biochemical purification and molecular characterization of the Drosophila COMPASS family. We observed a one-to-one similarity in subunit composition with their mammalian counterparts, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. Our study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila.     

E2F1 mediates DNA damage and apoptosis through HCF‐1 and the MLL family of histone methyltransferases

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti‐oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1‐to‐S phase transition, E2F1 associates through a short DHQY sequence with the cell‐cycle regulator HCF‐1 together with the mixed‐lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF‐1‐binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF‐1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF‐1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF‐1‐binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF‐1 association with E2F1 is a regulator of E2F1‐induced apoptosis.     

Cleavage of growth hormone by rabbit liver plasmalemma enhances binding

Several studies have shown that proteolytic cleavage can enhance the biological activity of the growth hormone (GH) molecule. It seemed possible, therefore, that proteolytic modification of GH might be a normal function of GH-target tissues. Plasmalemma-enriched fractions isolated from rabbit liver were found to contain a proteinase(s) which cleaves the large disulfide loop of human and rat GH. The proteolytic activity was specific to plasmalemma-enriched fractions in that much lower activities were observed in microsomal-enriched fractions prepared from the same livers. The plasmalemmal proteinase(s) may be a trypsin-like enzyme because proteolytic activity was decreased by two serine proteinase inhibitors. Inhibition by unlabeled human GH of 125I-GH binding to receptors did not prevent cleavage of the tracer; therefore, hormone-receptor interaction was not required for cleavage of the GH molecule. In binding studies, cleaved GH associated more readily than did intact hormone with rabbit liver receptors. These studies suggest that plasmalemma-enriched fractions prepared from rabbit liver contain a proteinase which cleaves the GH molecule in a highly specific manner. Moreover, it is unlikely that inactivation of GH is the function of this limited proteolysis because cleaved hormone is bound preferentially by at least a subset of receptors in rabbit liver.