MAP4K4 Is a Threonine Kinase That Phosphorylates FARP1

Mitogen-activated protein kinase
4 (MAP4K4) regulates the MEK kinase
cascade and is implicated in cytoskeletal rearrangement and migration;
however, identifying MAP4K4 substrates has remained a challenge. To
ascertain MAP4K4-dependent phosphorylation events, we combined phosphoproteomic
studies of MAP4K4 inhibition with in vitro assessment
of its kinase specificity. We identified 235 phosphosites affected
by MAP4K4 inhibition in cells and found that pTP and pSP motifs were
predominant among them. In contrast, in vitro assessment
of kinase specificity showed that MAP4K4 favors a pTL motif. We showed
that MAP4K4 directly phosphorylates and coimmunoprecipitates with
FERM, RhoGEF, and pleckstrin domain-containing protein 1 (FARP1).
MAP4K4 inhibition in SH-SY5Y cells increases neurite outgrowth, a
process known to involve FARP1. As FARP1 and MAP4K4 both contribute
to cytoskeletal rearrangement, the results suggest that MAP4K4 exerts
some of its effects on the cytoskeleton via phosphorylation of FARP1.