Conserved Glu40 and Glu433 of the biotin carboxylase domain of yeast pyruvate carboxylase I isoenzyme are essential for the association of tetramers.

The native form of pyruvate carboxylase is an alpha4 tetramer but the tetramerisation domain of each subunit is currently unknown. To identify this domain we co-expressed yeast pyruvate carboxylase 1 isozyme (Pyc1) with an N-terminal myc tag, together with constructs encoding either the biotin carboxylase (BC) domain or the transcarboxylase-biotin carboxyl carrier domain (TC-BCC), each with an N-terminal 9-histidine tag. From tag-affinity chromatography experiments, the subunit contacts within the tetramer were identified to be primarily located in the 55 kDa BC domain. From modelling studies based on known structures of biotin carboxylase domains and subunits we have predicted that Arg36 and Glu433 and Glu40 and Lys426, respectively, are involved pairwise in subunit interactions and are located on opposing subunits in the putative subunit interface of Pyc1. Co-expression of mutant forms with wild type Pyc1 showed that the R36E mutation had no effect on the interaction of these subunits with those of wild type Pyc1, while the E40R, E433R and R36E:E433R mutations caused severe loss of interaction with wild type Pyc1. Ultracentrifugal analysis of these mutants when expressed and purified separately indicated that the predominant form of E40R, E433R and R36R:E433R mutants is the monomer, and that their specific activities are less than 2% of the wild type. Studies on the association state and specific activity of the R36E mutant at different concentrations showed it to be much more susceptible to tetramer dissociation and inactivation than the wild type. Our results suggest that Glu40 and Glu433 play essential roles in subunit interactions.

Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively spliced exon 11 sequence to ligand binding and receptor activation.

The IR (insulin receptor) and IGFR (type I insulin-like growth factor receptor) are found as homodimers, but the respective pro-receptors can also heterodimerize to form insulin-IGF hybrid receptors. There are conflicting data on the ligand affinity of hybrids, and especially on the influence of different IR isoforms. To investigate further the contribution of individual ligand binding epitopes to affinity and specificity in the IR/IGFR family, we generated hybrids incorporating both IR isoforms (A and B) and IR/IGFR domain-swap chimaeras, by ectopic co-expression of receptor constructs in Chinese hamster ovary cells, and studied ligand binding using both radioligand competition and bioluminescence resonance energy transfer assays. We found that IR-A-IGFR and IR-B-IGFR hybrids bound insulin with similar relatively low affinity, which was intermediate between that of homodimeric IR and homodimeric IGFR. However, both IR-A-IGFR and IR-B-IGFR hybrids bound IGF-I and IGF-II with high affinity, at a level comparable with homodimeric IGFR. Incorporation of a significant fraction of either IR-A or IR-B into hybrids resulted in abrogation of insulin- but not IGF-I-stimulated autophosphorylation. We conclude that the sequence of 12 amino acids encoded by exon 11 of the IR gene has little or no effect on ligand binding and activation of IR-IGFR hybrids, and that hybrid receptors bind IGFs but not insulin at physiological concentrations regardless of the IR isoform they contained. To reconstitute high affinity insulin binding within a hybrid receptor, chimaeras in which the IGFR L1 or L2 domains had been replaced by equivalent IR domains were co-expressed with full-length IR-A or IR-B. In the context of an IR-A-IGFR hybrid, replacement of IR residues 325-524 (containing the L2 domain and part of the first fibronectin domain) with the corresponding IGFR sequence increased the affinity for insulin by 20-fold. We conclude that the L2 and/or first fibronectin domains of IR contribute in trans with the L1 domain to create a high affinity insulin-binding site within a dimeric receptor.

Regulation of mitotic entry by microcephalin and its overlap with ATR signalling.

Ataxia-telangiectasia mutated and Rad3 related (ATR)-Seckel syndrome and autosomal recessive primary microcephaly (MCPH) syndrome share clinical features. RNA interference (RNAi) of MCPH1 have implicated the protein it encodes as a DNA-damage response protein that regulates the transcription of Chk1 and BRCA1, two genes involved in the response to DNA damage. Here, we report that truncating mutations observed in MCPH-syndrome patients do not impact on Chk1 or BRCA1 expression or early ATR-dependent damage-induced phosphorylation events. However, like ATR-Seckel syndrome cells, MCPH1-mutant cell lines show defective G2-M checkpoint arrest and nuclear fragmentation after DNA damage, and contain supernumerary mitotic centrosomes. MCPH1-mutant and ATR-Seckel cells also show impaired degradation of Cdc25A and fail to inhibit Cdc45 loading onto chromatin after replication arrest. Additionally, microcephalin interacts with Chk1. We conclude that MCPH1 has a function downstream of Chk1 in the ATR-signalling pathway. In contrast with ATR-Seckel syndrome cells, MCPH1-mutant cells have low levels of Tyr 15-phosphorylated Cdk1 (pY15-Cdk1) in S and G2 phases, which correlates with an elevated frequency of G2-like cells displaying premature chromosome condensation (PCC). Thus, MCPH1 also has an ATR-independent role in maintaining inhibitory Cdk1 phosphorylation, which prevents premature entry into mitosis.