Determinants that control the distinct subcellular localization of p38α-PRAK and p38β-PRAK complexes

p38α and p38β MAPKs (mitogen-activated protein kinases) share about 80% of their protein sequence identity, but have quite different biological functions. One such difference is in regulating the subcellular localization of their downstream kinases, such as PRAK (p38-regulated/activated protein kinase or MK5). The p38α-PRAK complex is found in the nucleus, whereas the p38β-PRAK complex is exclusively localized to the cytosol. By generating a series of chimeric and point mutants of p38α and p38β, we found two amino acid residues (Asp¹⁴⁵ and Leu¹⁵⁶ in p38α, Gly¹⁴⁵ and Val¹⁵⁶ in p38β) that determine the distinct subcellular locations of p38α-PRAK and p38β-PRAK. The subcellular localization of MK2 (MAPK-activated protein kinase 2), another downstream kinase of p38, was regulated in the same manner as that of PRAK. We found that nuclear import, but not export, determines the subcellular localization of p38α-PRAK and p38β-PRAK. The published structure of the p38α-MK2 complex suggests Leu¹⁵⁶ of p38α is involved in the interaction with the nuclear localization signal in PRAK. The difference at this residue between p38α and p38β may affect the nuclear localization signal in PRAK differently, and thereby influence the import of the complexes. Asp¹⁴⁵ in p38α (or Gly¹⁴⁵ in p38β) is located on a different surface patch, and further random mutagenesis revealed that mutation of Asp¹⁴⁵, Thr¹²³, and Gln³²⁵, the residues that can directly interact with importin α as predicted by modeling, but not mutation of the other 7 amino acid residues that cannot reach importin α, re-locate p38α-PRAK to the cytosol, suggesting that interaction with import machinery is involved in determining the subcellular localization of the p38α-PRAK and p38β-PRAK complexes. Last, we show that nuclear localization of PRAK is required for its role in inhibiting the proliferation of NIH3T3 cells. In conclusion, multiple determinants control the distinct subcellular localization of p38α-PRAK and p38β-PRAK complexes, and the location of PRAK plays a role in its function.