Rationale MAP4K4 is a member of the GCKIV subfamily of the evolutionarily-conserved Ste20 kinases. GCKIVs possess an N-terminal catalytic domain and a C-terminal citron homology domain, joined by an extended disordered linker region. Here, we assess expression and regulation of MAP4K4 in cardiomyocytes.
Methods Neonatal and adult rat cardiomyocytes were used for transcriptomics and proteomics. Plasmids or adenoviruses containing FLAG-MAP4K4 were prepared for transfection or infection into HEK209T cells or cardiomyocytes, respectively. Cell extracts were examined using immunoprecipitation and immunoblotting.
Results Bioinformatics analysis showed that the rat MAP4K4 gene comprises 33 exons encoding a 1393-residue maximum-length protein. A MAPK4K4 transcript encoding a 1233-residue protein was cloned from cardiomyocytes. Compared with the 1178-residue rat MAP4K4 in the NCBI database (NP_001100374.1), MAP4K4 (1–1233) possessed a 55-residue N-terminal extension containing the ATP-binding site. As in humans, rat MAP4K4 was extensively alternatively-spliced in the linker region. Using proteomics, MAP4K4 ranked 5th amongst the 28 Ste20 kinases in relative abundance. Inhibition of PP2A by calyculin A promoted multisite phosphorylations (confirmed by treatment with exogenous PP2A) of FLAG-MAP4K4 (1–1233). Kinase-dead FLAG-MAP4K4 (T187A) or the FLAG-MAP4K4 (1–307) catalytic domain were not hyperphosphorylated with calyculin A, indicating that MAP4K4 autophosphorylates its activation loop Thr-187, promoting additional autophosphorylations C-terminal to the catalytic domain. Deletion analysis indicated these phosphorylation sites lie within the N-terminal half of the linker.
Conclusions MAP4K4 is a highly-expressed kinase in cardiomyocytes that exists as multiple isoforms. Its activity is probably restrained by proximity to PP2A and it most likely plays a significant role as an apex kinase in cardiomyocyte biochemistry.
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