PSTPIP2 tyrosine phosphorylation and interaction with membrane phospholipids are required for PSTPIP2 inhibition of osteoclast differentiation. (A) Predicted structural determinants of PSTPIP2 molecular interactions. (B) Mutation of the conserved cationic residues, R116 and K117 (KRQ), abolishes phospholipid binding. (C) Mutation of W232 to alanine (WA) inhibits PSTPIP2 interaction with PTP-PEST. (D) Mutation of the major tyrosine phosphorylation sites Y323 and Y333 (Y2F) eliminates tyrosine phosphorylation of PSTPIP2 in response to CSF-1 stimulation (middle panel), which is also reduced in CSF-1–stimulated macrophages expressing the KRQ mutant (right panel). Repositioned gel lanes from the same blot are separated by vertical lines. (E) The expression of WT PSTPIP2 and PSTPIP2 mutants was confirmed by Western blotting of SDS cell lysates. Actin indicates loading control. (F) Morphology of day 6 osteoclasts obtained from immortalized cmo c-Kit⁺ Mac-1^lo c-Fms^lo EOCP retrovirally transduced with vector (Vect), WT PSTPIP2 (P2WT), or the PSTPIP2 mutants described in panels B through E. (G) Quantitation of the number of OCLC per field. (H) Effects of PSTPIP2 deficiency and mutation on TRAP expression. AFU DAPI indicates arbitrary fluorescence units of DAPI-stained cultures. Data ± SEM, n ≥ 3 independent experiments; ns indicates not significant (P > .05).