Fig. S8.

Phosphorylation of Ser-22 may reduce D.d. V-1’s affinity for CP. (A and B) Two views of the structure of mouse V-1 (23) that provide insight into the possible effect Ser-22 phosphorylation might have on the function of D.d. V-1. (A) Loop 1 in mouse V-1 is critical for its interaction with CP, as mutation of the four residues marked (E33, G34, G35, and R36) results in a ∼200-fold decrease in V-1’s affinity for CP (27). Data presented here shows that loop 1 is also critical for the interaction of D.d. V-1 with D.d. CP. (A and B) In mouse V-1, Tyr-21 (Y21) sits in the position occupied by Ser-22 in D.d. V-1 and is likely hydrogen-bonded to Arg-30 (R30), thereby stabilizing the conformation of loop 1. Importantly, the overall folding of this region is probably conserved between mouse V-1 and D.d. V-1 because the hydrophobic residues involved in packing required to stabilize ankyrin repeat 1 (residues 5–23) and ankyrin repeat II (residues 39–57) are conserved. Given this, the introduction of a phosphate group on Ser-22 in D.d. V-1 would likely disrupt the conformation of loop 1, resulting in a decrease in the protein’s affinity for CP. (C) Alignment of the amino acid sequence of Dictyostelium V-1 with the amino acid sequences of human, mouse, rat, and chicken V-1 showing that these vertebrate V-1s contain a potentially phosphorylatable tyrosine (red box) at the position analogous to Ser-22 in D.d. V-1. Note that this implies that different kinases/signaling pathways are regulating V-1 in vertebrate cells and Dictyostelium. Interestingly, while Kitizawa et al. (50) presented indirect evidence that PKA is involved in the phosphorylation of mammalian V-1, precedents exist for the phosphorylation of tyrosines downstream of PKA (55).