Protein tyrosine phosphatase 1B undergoes mitosis-specific phosphorylation on serine.
We have investigated the regulation of protein tyrosine phosphatase 1B (PTP1B) through the cell cycle of HeLa cells. PTP1B from HeLa cells arrested in mitosis migrated more slowly during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than did PTP1B from unsynchronized HeLa cells. To explore whether this mobility shift was caused by phosphorylation, PTP1B was immunoprecipitated from 32Pi-labeled unsynchronized and mitotic HeLa cells. PTP1B from mitotic cells incorporated significantly more 32Pi than did PTP1B from unsynchronized cells. Alkaline phosphatase treatment of mitotic HeLa cell lysates resulted in the conversion of PTP1B to its more rapidly migrating form, confirming that the mobility shift was a result of the mitotic phosphorylation. Phosphoamino acid analysis of PTP1B from mitotic cells revealed that PTP1B became phosphorylated on serine. Dephosphorylation of PTP1B occurred following the release of cells from nocodazole synchronization and was independent of new protein synthesis. This dephosphorylation was inhibited by okadaic acid, a potent inhibitor of types 1 and 2A serine/threonine phosphatases. The mitotic phosphorylation had no apparent effect on the activity of PTP1B as measured in in vitro phosphatase assays using 32P-labeled Raytide as substrate. p34cdc2 appears not to be the mitotic PTP1B kinase, as mapping experiments showed that this enzyme phosphorylated PTP1B on a site different from that on which it was phosphorylated in vivo. These observations suggest that PTP1B may be differentially regulated through the cell cycle.
Schievella
A R
AR
Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138.
Paige
L A
LA
Johnson
K A
KA
Hill
D E
DE
Erikson
R L
RL
D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D004988:Ethers, Cyclic / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000502:physiology*; D019319:Okadaic Acid; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*
D004988:Ethers, Cyclic; D019319:Okadaic Acid; D012694:Serine; D016203:CDC2 Protein Kinase; D017027:Protein Tyrosine Phosphatases