The T-cell protein tyrosine phosphatase is phosphorylated on Ser-304 by cyclin-dependent protein kinases in mitosis.

Two alternatively spliced forms of the human protein tyrosine phosphatase TCPTP (T-cell protein tyrosine phosphatase) exist: a 48 kDa form that is targeted to the endoplasmic reticulum (TC48) and a shorter 45 kDa form that is targeted to the nucleus (TC45). In this study we have identified Ser-304 (Phe301-Asp-His-Ser304-Pro-Asn-Lys307) as a major TCPTP phosphory-lation site and demonstrate that TC45, but not TC48, is phosphorylated on this site in vivo. Phosphorylation of TC45 on Ser-304 was cell cycle-dependent, and increased as cells progressed from G2 into mitosis, but subsided upon mitotic exit. Ser-304 phosphorylation was increased when cells were arrested in mitosis by microtubule poisons such as nocodazole, but remained unaltered when cells were arrested at the G2/M checkpoint by adriamycin. Phosphorylation of Ser-304 did not alter significantly the phosphatase activity or the protein stability of TC45, and had no apparent effect on TC45 localization. Ser-304 phosphorylation was ablated when cells were treated with the CDK (cyclin-dependent protein kinase) inhibitors roscovitine or SU9516, but remained unaltered when ERK1/2 activation was inhibited with the MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase) inhibitor PD98059. In addition, recombinant CDKs, but not the Polo-like kinase Plk1, phosphorylated Ser-304 in vitro. Our studies identify Ser-304 as a major phosphorylation site in human TCPTP, and the TC45 variant as a novel mitotic CDK substrate.

Nuclear transport of parathyroid hormone (PTH)-related protein is dependent on microtubules.

PTH-related protein (PTHrP) was first discovered as a circulating factor secreted by certain cancers and is responsible for the syndrome of humoral hypercalcemia of malignancy induced by various tumors. The similarity of its N terminus to that of PTH enables PTHrP to share the signaling properties of PTH, but the rest of the molecule possesses distinct functions, including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import is mediated by importin beta1. In this study we use the technique of fluorescence recovery after photobleaching to demonstrate the ability of PTHrP to shuttle between cytoplasm and nucleus and to visualize directly the transport of PTHrP into the nucleus in living cells. Endogenous and transfected PTHrP was demonstrated to colocalize with microtubule structures in situ using various high-resolution microscopic approaches, as well as in in vitro binding studies, where importin beta1, but not importin alpha, enhanced the microtubular association of PTHrP with microtubules. Significantly, the dependence of PTHrP nuclear import on microtubules was shown by the inhibitory effect of pretreatment with the microtubule-disrupting agent nocodazole on nuclear-cytoplasmic flux. These results indicate that PTHrP nuclear/nucleolar import is dependent on microtubule integrity and are consistent with a direct role for the cytoskeleton in protein transport to the nucleus.