Generation of CKAR. (A) CKAR is comprised of mCFP, the FHA2 domain of Rad53p, a PKC substrate sequence, and mYFP. The substrate sequence, when phosphorylated, binds the FHA2 phospho-peptide–binding domain. This conformational change results in a change in FRET, reversible by phosphatases. (B) CKAR is stoichiometrically phosphorylated by PKC in vitro. Time course of ³²P incorporation into nickel-purified histidine-tagged CKAR measured by scintillation counts of excised Coomassie blue–stained bands. (Inset) Coomassie blue–stained purified CKAR (top) and ³²P autoradiography (bottom). (C) Emission spectra of CKAR incubated for 30 min at 30°C with and without purified PKCβII. Excitation at 434 nm resulted in a CFP emission peak (476 nm) and YFP emission peak caused by FRET from CFP (528 nm). PKC phosphorylation resulted in decreased intensity at 528 nm and increased intensity at 476 nm, consistent with a decrease in FRET. Incubation of CKAR with trypsin for 30 min at 30°C destroyed the YFP emission, demonstrating that FRET was caused by intramolecular energy transfer. (D) Incubation with active calmodulin-dependent kinase II (CaMKII) or cAMP-dependent kinase (PKA) resulted in no change in FRET.

Targeting CKAR to plasma membrane. (A) CKAR was targeted to plasma membrane by fusion of the 10 amino acid NH₂ terminus of the kinase Lyn to the NH₂ terminus of CKAR, encoding myristoylation and palmitoylation. (Inset) An image of MyrPalm-CKAR expressed in HeLa cells showing effective targeting of CKAR to the plasma membrane. (B) Supramaximal stimulation of PKC with 200 nM PDBu results in nearly complete phosphorylation of CKAR, since inhibition of phosphatases with 100 nM calyculin A results in only slight additional phosphorylation. (C) MyrPalm-CKAR phosphorylation oscillates after 10 μM histamine and is inhibited by 1 μM Gö6983. (D) Expanded time scale of Fig. 3 C. (E) Pretreatment with 1 μM Gö6983 prevents MyrPalm-CKAR phosphorylation by 10 μM histamine. (F) Mutation of the threonine phosphoacceptor to alanine (T413A) makes MyrPalm-CKAR unresponsive to 10 μM histamine. All data are representative of at least three experiments. Oscillatory phosphorylation, while highly variable, was detected in 10–20% of cells studied, observed in over 30 cells in more than 12 different experiments.

Histamine induces oscillations of PKC translocation. (A) YFP fused to both termini of PKC functions as a FRET acceptor for CFP fused to the myristoylation and palmitoylation sequence from Lyn (MyrPalm-CFP). Translocation of YFP-PKC-YFP to a membrane containing MyrPalm-CFP results in increased FRET. (B) Addition of 10 μM histamine to HeLa cells causes rapid, oscillating translocation of YFP-PKC-YFP to plasma membrane (black) corresponding to oscillating calcium transients imaged by Fura red in the same cell (red). Data are representative of three experiments.

PLC controls oscillations of PKC activity in MDCK cells. 10 μM ATP induces calcium transients phase locked with responses of MyrPalm-CKAR (reports phosphorylation) (A), CYPHR (reports PLC activity) (B), and DAGR (reports DAG) (C). All data are representative of three to five experiments.

CKAR is a specific, reversible reporter for PKC activation in live cells. (A) CKAR expressed in HeLa cells is phosphorylated upon stimulation of PKC with 200 nM PDBu. This is reversed by addition of 500 nM Go6983, a specific inhibitor of PKC. (B) CKAR phosphorylation is specific for PKC. (Black) PKC is activated by 200 nM PDBu and inhibited by 1 μM Go6983. (Red) Neither DMSO (vehicle for PDBu) nor 10 μM forskolin cause a change in FRET. (Green) Preinhibition of PKC (1 μM Go6983) does not change basal FRET, which remains unchanged by release of intracellular calcium by thapsigargin to stimulate other calcium-sensitive kinases such as CaMKII. (C) Supramaximal stimulation of PKC with 200 nM PDBu results in stable phosphorylation of CKAR, but inhibition of phosphatases with 100 nM calyculin A results in additional phosphorylation. Data are from two cells in the same field of view. (D) Phosphorylation of CKAR in the cytosol (red) and nucleus (black) reveal preferential cytosolic phosphorylation after PDBu but greater and uniform phosphorylation after calyculin A treatment. (E) Images corresponding to Fig. 2 D show phosphorylation (red shift of pseudocolored FRET ratio image, top) of CKAR after PDBu (20') and PDBu and calyculin (40'). YFP intensity images (bottom) indicate no change in CKAR localization over the course of the experiment. (F) Mutation of the threonine phosphoacceptor in the designed PKC substrate of CKAR precludes FRET changes in response to either 200 nM PDBu or 100 nM calyculin. (G) CKAR is also sensitive to receptor-mediated activation of PKC. 10 μM histamine resulted in rapid phosphorylation of CKAR in HeLa cells. All data are representative of at least three experiments.

MyrPalm-CKAR oscillatory phosphorylation corresponds to calcium oscillations. (A) Calcium (Fura red intensity, red) and MyrPalm-CKAR phosphorylation (CFP-YFP FRET, black) show that MyrPalm-CKAR phosphorylation corresponds directly to calcium transients. (B) Averaging the calcium and phosphorylation peaks in A illustrates a consistent lag of 10–20 s in MyrPalm-CKAR phosphorylation after initiation of calcium transients. The time of each Fura red intensity spike was normalized and the Fura red intensities and FRET ratios averaged for each image acquisition surrounding that fixed time. (C) Histamine stimulation of HeLa cells expressing MyrPalm-CKAR T413A shows that FRET changes are almost entirely independent of spectral overlap from Fura red signals and instead depend on the phosphoacceptor T413 in the PKC substrate of MyrPalm-CKAR. (D) Histamine stimulation resulting in calcium oscillations does not result in oscillatory phosphorylation of cytosolic CKAR. All data are representative of at least three experiments. Phase-locked calcium and phosphorylation oscillations have been noted in 15 different cells from eight experiments.

Translocation of the PH domain of PLCδ reported by FRET reveals constant PLC activity during calcium oscillations. (A) CYPHR, a CFP-PHδ-YFP fusion construct, reports PLC activity by reduced intermolecular FRET upon translocation from membrane. The PH domain of PLCδ1, which binds PIP₂ in the plasma membrane, translocates to cytosol upon PIP₂ hydrolysis. Translocation from membrane (two dimensions) to cytosol (three dimensions) results in a decreased effective concentration and hence lower intermolecular FRET. (B) CYPHR reports constant PLC activity (black) during calcium oscillations (red). Data are representative of four experiments.

A model of the temporal control of PKC signals. Inactive, cytosolic PKC is autoinhibited by a pseudosubstrate (1). Phospholipase activation results in DAG formation and calcium release: calcium binds cytosolic PKC (2), imparting weak membrane affinity (3). Calcium-bound PKC rapidly binds DAG in the membrane, resulting in maximal membrane affinity and full PKC activity by removal of the pseudosubstrate from the active site (4). A drop in either calcium or DG results in decreased membrane affinity and reinhibition by the pseudosubstrate (5). This coincidence detection by PKC defines a signaling nexus in the cell, whereby strict temporal fidelity of phosphorylation is dictated by rapid turnover of either second messenger and counterbalancing phosphatase activity.