Intraperitoneal injection of epidermal growth factor (EGF) into mice resulted in the phosphorylation of liver nuclei phospholipase Cgamma1 (PLCgamma1) at the tyrosine, coincident with the time course of nuclear membrane epidermal growth factor receptor (EGFR) activation. The function of PLCgamma1 in mice liver nuclei was attributed to a 120 kDa protein fragment. This 120 kDa protein was immunoprecipitated with the isozyme specific PLCgamma1 antibody and was found to be sensitive to a PLCgamma1 specific blocking peptide. The 10-partial sequence analysis revealed that the 120 kDa protein contains the PELCQVSLSE sequence at its N-terminal end and the RTRVNGDNRL sequence at its C-terminal end, which reveals that this protein is a major fragment of PLCgamma1 devoid of an amino acid portion at the N-terminal end. The tyrosine-phosphorylated 120 kDa protein interacts with activated EGFR, binds phosphatidylinositol-3-OH-kinase enhancer (PIKE), enhances nuclear phosphatidylinositol-3-OH-kinase (PI[3]K) activity, and generates diacylglycerol (DAG) in response to the EGF signal to the nucleus in vivo. The immunoprecipitated 120 kDa protein fragment displayed phosphatidylinositol (PI) hydrolysis activity. These results establish the capacity of EGF-triggered nuclear signaling which is mediated by EGFR itself, located on the inner nuclear membrane. This is the first report identifying a 120 kDa PLCgamma1 fragment generated in vivo in the nucleus and capable of discharging the function of nuclear PLCgamma1.
There is growing evidence for the role of protein tyrosine phosphatases in controlling such fundamental cellular processes as growth and differentiation. Pervanadate is a potent inhibitor of protein tyrosine phosphatase which has been observed here to induce proliferation in C3H10T1/2 mouse fibroblasts. Pervanadate also translocated/activated p42/44 mitogen-activated protein (MAP) kinase to the cell nucleus. An almost similar pattern of nuclear p42/44 MAP kinase stimulation is seen with TPA. On the other hand, TPA treatment results in a rapid activation of cytosolic MAP kinase which declines with time. Thus pervanadate appears as a very useful tool for studying tyrosine phosphorylation.
A Ca 2؉ -pump ATPase, similar to that in the endoplasmic reticulum, has been located on the outer membrane of rat liver nuclei. The effect of cAMP-dependent protein kinase (PKA) on nuclear Ca 2؉ -ATPase (NCA) was studied by using purified rat liver nuclei. Treatment of isolated nuclei with the catalytic unit of PKA resulted in the phosphorylation of a 105-kDa band that was recognized by antibodies specific for sarcoplasmic reticulum Ca 2؉ -ATPase type 2b. Partial purification and immunoblotting confirmed that the 105-kDa protein band phosphorylated by PKA is NCA. The stoichiometry of phosphorylation was 0.76 mol of phosphate incorporated͞mol of partially purified enzyme. Measurement of ATP-dependent 45 Ca 2؉ uptake into purified nuclei showed that PKA phosphorylation enhanced the Ca 2؉ -pumping activity of NCA. We show that PKA phosphorylation of Ca 2؉ -ATPase enhances the transport of 10-kDa fluorescent-labeled dextrans across the nuclear envelope. The findings reported in this paper are consistent with the notion that the crosstalk between the cAMP͞PKA-and Ca 2؉ -dependent signaling pathways identified at the cytoplasmic level extends to the nucleus. Furthermore, these data support a function for crosstalk in the regulation of calcium-dependent transport across the nuclear envelope.
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