Portland Press

Figure 1.

$(A) The predicted first exon and start codon for the rat MAP4K4 gene were identified by 5′-RACE using rat heart mRNA. The amplified product was cloned into a pDRIVE vector used to transform DH5α cells. Clones were screened by PCR using a primer from the 3′ end of the first exon (upper panel; individual clones numbered 1–6). Clone 6 was sequenced (lower panel) and contained the start codon (red box) plus upstream non-coding sequence. (B) The predicted protein-coding sequence of rat MAP4K4 cloned from neonatal rat cardiomyocytes is shown, identifying the kinase domain (green), the citron homology domain (blue), the region subject to alternative splicing (exons 15–25; red italics) and the region containing a predicted nuclear-localisation signal (black, bold type; key residues identified by NucPred outlined in black). (C) Evidence for alternatively spliced MAP4K4 mRNAs in rat cardiomyocytes with a schematic of the spliced exons and positions of PCR primers (upper panel; F, forward primer; R, reverse primer). (i) cDNAs were cloned from myocytes with and without exon 21 (identified by DNA sequencing). This was confirmed using primers spanning the exon boundary. Primers F1 and R5 were used for PCR to amplify MAP4K4 sequences between exons 14 and 19 (ii) and primers F2 and R6 were used to amplify MAP4K4 sequences between exons 22 and 26 (iii). Multiple bands were detected in cDNAs prepared for RACE and from cardiomyocyte (CM) RNA, consistent with alternative splicing across the region. (D) Cardiomyocyte MAP4K4 is expressed in the cytosol and in high salt extracts, enriched for nuclear proteins. Cardiomyocyte proteins (cytosol: 0.2 × 106 cells; nuclear-enriched high salt extracts: 0.4 × 106 cells) were immunoblotted for MAP4K4 (8% (w/v) polyacrylamide gels, electrophoresis: 60 min, 200 V; upper immunoblots), Gapdh (10% (w/v) polyacrylamide gels; lower left immunoblot) or CREB (10% (w/v) polyacrylamide gels; lower right immunoblot). The assessment was conducted on 6 occasions with independent cardiomyocyte preparations. Although MAP4K4 was routinely detected in the high salt extracts, there was variation and blots show the experiment with the least amount of MAP4K4 detected in this fraction (left panels). Densitometric analysis of each of the experiments is provided (right panel). The proportion of MAP4K4 was calculated on a per cell basis, allowing for the volume of extracts loaded onto the gel (10 µl for each lane for the blots shown) relative to the total volumes of each of the cytosol (200 µl) and nuclear-enriched high salt extracts (70 µl) containing proteins from 4 × 106 cells. Individual data points are shown with means ± SEM (n = 6).$

Characterisation of rat MAP4K4.

(A) The predicted first exon and start codon for the rat MAP4K4 gene were identified by 5′-RACE using rat heart mRNA. The amplified product was cloned into a pDRIVE vector used to transform DH5α cells. Clones were screened by PCR using a primer from the 3′ end of the first exon (upper panel; individual clones numbered 1–6). Clone 6 was sequenced (lower panel) and contained the start codon (red box) plus upstream non-coding sequence. (B) The predicted protein-coding sequence of rat MAP4K4 cloned from neonatal rat cardiomyocytes is shown, identifying the kinase domain (green), the citron homology domain (blue), the region subject to alternative splicing (exons 15–25; red italics) and the region containing a predicted nuclear-localisation signal (black, bold type; key residues identified by NucPred outlined in black). (C) Evidence for alternatively spliced MAP4K4 mRNAs in rat cardiomyocytes with a schematic of the spliced exons and positions of PCR primers (upper panel; F, forward primer; R, reverse primer). (i) cDNAs were cloned from myocytes with and without exon 21 (identified by DNA sequencing). This was confirmed using primers spanning the exon boundary. Primers F1 and R5 were used for PCR to amplify MAP4K4 sequences between exons 14 and 19 (ii) and primers F2 and R6 were used to amplify MAP4K4 sequences between exons 22 and 26 (iii). Multiple bands were detected in cDNAs prepared for RACE and from cardiomyocyte (CM) RNA, consistent with alternative splicing across the region. (D) Cardiomyocyte MAP4K4 is expressed in the cytosol and in high salt extracts, enriched for nuclear proteins. Cardiomyocyte proteins (cytosol: 0.2 × 10⁶ cells; nuclear-enriched high salt extracts: 0.4 × 10⁶ cells) were immunoblotted for MAP4K4 (8% (w/v) polyacrylamide gels, electrophoresis: 60 min, 200 V; upper immunoblots), Gapdh (10% (w/v) polyacrylamide gels; lower left immunoblot) or CREB (10% (w/v) polyacrylamide gels; lower right immunoblot). The assessment was conducted on 6 occasions with independent cardiomyocyte preparations. Although MAP4K4 was routinely detected in the high salt extracts, there was variation and blots show the experiment with the least amount of MAP4K4 detected in this fraction (left panels). Densitometric analysis of each of the experiments is provided (right panel). The proportion of MAP4K4 was calculated on a per cell basis, allowing for the volume of extracts loaded onto the gel (10 µl for each lane for the blots shown) relative to the total volumes of each of the cytosol (200 µl) and nuclear-enriched high salt extracts (70 µl) containing proteins from 4 × 10⁶ cells. Individual data points are shown with means ± SEM (n = 6).

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