Biochem. J. (2007) volume 406, issue 3, pages 389–398, https://doi.org/10.1042/BJ20070091

It has come to the attention of the authors that in the original Figure 2(D) the same images were used for GST-CK1δ1-428, GST-CK1δ1-428S370A and GST-CK1δ1-375 in the Coomassie-stained gel.

In the autoradiogram of Figure 2(D), PKA negative GST-CK1δ1-428, and PKA negative GST-CK1δ1-428S370A were the same, as were GST-CK1δ1-350 and GST-CK1δ1-375. This mistake has now been addressed and corrected.

In Figure 3(A) fraction number 14 of the control was accidentally used for fraction number 14 of H89, degeulin, and calphostin treatments. In addition, fraction number 13 of the control was erroneously used for fraction 13 in the deguelin treatment. Furthermore, fraction number 9 of the deguelin treatment was run on another gel, and the image was subsequently inserted into the Figure.

The authors are now presenting the corrected Figures 2(D) and 3(A), and would like to apologize for any confusion that these errors may have caused. The new Figures have been assessed and approved by the Editorial Board of the Biochemical Journal.

New Figure 2(D)

Phosphorylation of GST-CK1δ fusion proteins by PKA.

Figure 2.(D)
Phosphorylation of GST-CK1δ fusion proteins by PKA.

In vitro kinase assays were performed using the catalytic subunit of PKA purified from porcine heart as source of enzyme and the following GST-CK1δ fusion proteins (FPs) as substrates: (i) GST-CK1δ1-428 (FP449), (ii) GST-CK1δ150-428 (FP894), (iii) GST-CK1δ1-428S370A (FP1023), (iv) GST-CK1δ52-304 (FP882), (v) GST-CK1δ1-350 (FP898) and (vi) GST-CK1δ1-375 (FP897). Equal amounts of GST-CK1δ fusion proteins were loaded on to 12% SDS-polyacrylamide gels and stained with Coomassie Blue. The phosphorylated proteins were detected following autoradiography of the dried Coomassie-stained gels. Quantification of phosphate incorporation of phosphorylated GST-CK1δ fusion proteins was measured by Cerenkov counting. GST-CK1δ fusion proteins containing the complete kinase domain were also phosphorylated in the absence of PKA due to autophosphorylation. However, phosphate incorporation into GST-CK1δ fusion proteins containing Ser370 increased when PKA was used as the enzyme in the in vitro kinase reactions. The dotted line indicates that the two last lanes aa1-375 correspond to a different gel.

Figure 2.(D)
Phosphorylation of GST-CK1δ fusion proteins by PKA.

In vitro kinase assays were performed using the catalytic subunit of PKA purified from porcine heart as source of enzyme and the following GST-CK1δ fusion proteins (FPs) as substrates: (i) GST-CK1δ1-428 (FP449), (ii) GST-CK1δ150-428 (FP894), (iii) GST-CK1δ1-428S370A (FP1023), (iv) GST-CK1δ52-304 (FP882), (v) GST-CK1δ1-350 (FP898) and (vi) GST-CK1δ1-375 (FP897). Equal amounts of GST-CK1δ fusion proteins were loaded on to 12% SDS-polyacrylamide gels and stained with Coomassie Blue. The phosphorylated proteins were detected following autoradiography of the dried Coomassie-stained gels. Quantification of phosphate incorporation of phosphorylated GST-CK1δ fusion proteins was measured by Cerenkov counting. GST-CK1δ fusion proteins containing the complete kinase domain were also phosphorylated in the absence of PKA due to autophosphorylation. However, phosphate incorporation into GST-CK1δ fusion proteins containing Ser370 increased when PKA was used as the enzyme in the in vitro kinase reactions. The dotted line indicates that the two last lanes aa1-375 correspond to a different gel.

New Figure 3(A)

PKA is the major CK1δCK activity present in the kinase peak fraction of fractionated MiaPaCa-2 cells.

Figure 3.
PKA is the major CK1δCK activity present in the kinase peak fraction of fractionated MiaPaCa-2 cells.

(A) CK1δ-targeting kinase activities are mainly inhibited by the PKA-specific inhibitor H89. In vitro kinase assays were performed in the absence and presence of the PKA-specific inhibitor H89 (5 μM), the Akt-specific inhibitor degeulin (200 nM) or the PKC-specific inhibitor calphostin C (1 μM), using GST–CK1δ305–375 (FP1006) as the substrate and fractions A7-B4 of fractionated MiaPaCa-2 cell lysates as sources of kinase activity. The phosphorylated proteins were detected following autoradiography of the dried Coomassie-stained gels. Quantification of phosphate incorporation of phosphorylated GST-CK1δ305-375 (FP1006) was measured by Cerenkov counting. [Note: data from an experiment performed in parallel in 2006 was used for this Figure]. Since the gel for calphostin was broken between lanes 13 and 14, the two pieces were brought more closely together which is indicated by the dotted line.

Figure 3.
PKA is the major CK1δCK activity present in the kinase peak fraction of fractionated MiaPaCa-2 cells.

(A) CK1δ-targeting kinase activities are mainly inhibited by the PKA-specific inhibitor H89. In vitro kinase assays were performed in the absence and presence of the PKA-specific inhibitor H89 (5 μM), the Akt-specific inhibitor degeulin (200 nM) or the PKC-specific inhibitor calphostin C (1 μM), using GST–CK1δ305–375 (FP1006) as the substrate and fractions A7-B4 of fractionated MiaPaCa-2 cell lysates as sources of kinase activity. The phosphorylated proteins were detected following autoradiography of the dried Coomassie-stained gels. Quantification of phosphate incorporation of phosphorylated GST-CK1δ305-375 (FP1006) was measured by Cerenkov counting. [Note: data from an experiment performed in parallel in 2006 was used for this Figure]. Since the gel for calphostin was broken between lanes 13 and 14, the two pieces were brought more closely together which is indicated by the dotted line.

To support data shown in Figure 3(A), results from experiments carried out in May 2017 are now presented:

PKA is the major CK1δ CK activity present in the kinase peak fraction of fractionated MiaPaCa-2 cells

Figure 3.
PKA is the major CK1δ CK activity present in the kinase peak fraction of fractionated MiaPaCa-2 cells

CK1δ-targeting kinase activities are mainly inhibited by the PKA-specific inhibitor H89. In vitro kinase assays were performed in the absence and presence of the PKA-specific inhibitor H89 (5 μM), or the Akt-specific inhibitor degeulin (200 nM), using GST–CK1δ305–375 (FP1006) as the substrate and fractions A9-B11 (corresponding to serial numbers 9 to 23) of fractionated MiaPaCa-2 cell lysates as sources of kinase activity. The phosphorylated proteins were detected following autoradiography of the dried Coomassie-stained gels. Quantification of phosphate incorporation of phosphorylated GST-CK1δ305-375 (FP1006) was measured by Cerenkov counting. [Note: data from an experiment performed in 2017 was used for this Figure].

Figure 3.
PKA is the major CK1δ CK activity present in the kinase peak fraction of fractionated MiaPaCa-2 cells

CK1δ-targeting kinase activities are mainly inhibited by the PKA-specific inhibitor H89. In vitro kinase assays were performed in the absence and presence of the PKA-specific inhibitor H89 (5 μM), or the Akt-specific inhibitor degeulin (200 nM), using GST–CK1δ305–375 (FP1006) as the substrate and fractions A9-B11 (corresponding to serial numbers 9 to 23) of fractionated MiaPaCa-2 cell lysates as sources of kinase activity. The phosphorylated proteins were detected following autoradiography of the dried Coomassie-stained gels. Quantification of phosphate incorporation of phosphorylated GST-CK1δ305-375 (FP1006) was measured by Cerenkov counting. [Note: data from an experiment performed in 2017 was used for this Figure].

Phosphate incorporation in GST-CK1δ305-375 (FP1006) in the absence or presence of deguelin at 0.2 and 1.0 µM

PKA is the major CK1δCK activity present in the kinase peak fraction of fractionated MiaPaCa-2 cells

Figure 3.
PKA is the major CK1δCK activity present in the kinase peak fraction of fractionated MiaPaCa-2 cells

In vitro kinase assays were performed in presence of the Akt-specific inhibitor degeulin (200 nM and 1 µM), using GST–CK1δ305–375 (FP1006) as the substrate and fractions A1-C4 (corresponding to serial numbers 9 to 28) of fractionated MiaPaCa-2 cell lysates as sources of kinase activity. The phosphorylated proteins were detected following autoradiography of the dried Coomassie-stained gels. Quantification of phosphate incorporation of phosphorylated GST-CK1δ305-375 (FP1006) was measured by Cerenkov counting. [Note: data from an experiment performed in 2017 was used for this Figure].

Figure 3.
PKA is the major CK1δCK activity present in the kinase peak fraction of fractionated MiaPaCa-2 cells

In vitro kinase assays were performed in presence of the Akt-specific inhibitor degeulin (200 nM and 1 µM), using GST–CK1δ305–375 (FP1006) as the substrate and fractions A1-C4 (corresponding to serial numbers 9 to 28) of fractionated MiaPaCa-2 cell lysates as sources of kinase activity. The phosphorylated proteins were detected following autoradiography of the dried Coomassie-stained gels. Quantification of phosphate incorporation of phosphorylated GST-CK1δ305-375 (FP1006) was measured by Cerenkov counting. [Note: data from an experiment performed in 2017 was used for this Figure].