The protein tyrosine phosphatase PTP-PEST is an 88 kDa cytosolic enzym
e which is ubiquitously expressed in mammalian tissues. We have expres
sed PTP-PEST using recombinant baculovirus, and purified the protein e
ssentially to homogeneity in order to investigate phosphorylation as a
potential mechanism of regulation of the enzyme. PTP-PEST is phosphor
ylated in vitro by both cyclic AMP-dependent protein kinase (PKA) and
protein kinase C (PKC) at two major sites, which we have identified as
Ser39 and Ser435. PTP-PEST is also phosphorylated on both Ser39 and S
er435 following treatment of intact HeLa cells with TPA, forskolin or
isobutyl methyl xanthine (IBMX). Phosphorylation of Ser39 in vitro dec
reases the activity of PTP-PEST by reducing its affinity for substrate
. In addition, PTP-PEST immunoprecipitated from TPA-treated cells disp
layed significantly lower PTP activity than enzyme obtained from untre
ated cells. Our results suggest that both PKC and PKA are capable of p
hosphorylating, and therefore inhibiting, PTP-PEST in vivo, offering a
mechanism whereby signal transduction pathways acting through either
PKA or PKC may directly influence cellular processes involving reversi
ble tyrosine phosphorylation.