Protein Kinase D2 Regulates GRASP65 Phosphorylation and Golgi Ribbon Unlinking During G2/M Transition

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Does PKD2 dependent phosphorylation of GRASP65 at S-274 affect, phosphorylation by JNK at S-277? is there a connection between these two phosphorylation sites by the distinct kinases.

 

Is S-274 phosphorylated GRASP65 at the Golgi apparatus? 

Is GRASP55 unaffected by PKD2

Author Response

We would like to thank the Reviewer for the time dedicated to evaluating our manuscript and for the constructive comments. We believe that addressing the Reviewers' concerns has improved the quality of the work. We have carefully addressed all the issues raised, and the changes made to the manuscript are highlighted in the revised version, which now contains an additional panel in Supplementary Figure 2C. 

R1  Does PKD2 dependent phosphorylation of GRASP65 at S-274 affect, phosphorylation by JNK at S-277? Is there a connection between these two phosphorylation sites by the distinct kinases?

Serine 277 in the rat sequence corresponds to Serine 274 in the human sequence; at position 274 in the rat, a Glycine is found instead of a Serine. Both Serine residues are phosphorylated by JNK2 during the G2 phase of the cell cycle (Cervigni et al; J Cell Sci, 2016). The sequence surrounding Serine 274 does not match the consensus sequence for the PKCμ/PKD family, LxRxxS/T (Nishikawa et al., J Biol Chem. 1997; 272(2)), arguing against direct phosphorylation of this site by PKD. We have no evidence of a feedback loop in which phospho-GRASP65 promotes JNK2 activation; rather, JNK2 is itself activated by direct phosphorylation by MAP2K4 (MKK4) and MAP2K7 (MKK7) (work in progress). Thus, it cannot be excluded that PKD phosphorylates additional sites on GRASP65, an interesting suggestion that we consider worthy of future investigation. Our model suggests that PKD induces JNK2 activation through upstream MAP kinase cascade components, and that active JNK2 then phosphorylates GRASP65 at Serine 274. This is now more clearly explained in the text (lines 585–590).

 

Is S-274 phosphorylated GRASP65 at the Golgi apparatus?

While a small fraction of GRASP65 is cytoplasmic, most of the protein is bound to GM130 at the cis-Golgi (Ward et al, , 2001; J. Cell Biol; 155:4). We believe that the phosphorylated form of GRASP65 localizes to the Golgi complex, as phospho-GRASP65 can be detected at the Golgi even in non-synchronized cells in which the ribbon remains intact and we have not detected an increase of the signal in the cytoplasm. Furthermore, in G2 and mitotic cells we observe colocalization between phospho-GRASP65 and GM130. Notably, transfection of EGFP-WT-GRASP65 leads to an increase in GRASP65 phosphorylation, likely owing to displacement of the endogenous GRASP65 from GM130.

This is now better explained in the text (lines 245-248)

 

Is GRASP55 unaffected by PKD2?

It has been previously shown by Kienzle et al. (2013) that PKD controls Golgi fragmentation in G2 through a RAF1–MEK1 pathway. Moreover, Feinstein & Linstedt (2007-2008) showed that ERK can phosphorylate GRASP55 to induce Golgi fragmentation. Thus, as ERK is the downstream target of MEK1, likely GRASP55 phosphorylation is also controlled by PKD, but a specific antibody against phosphorylated GRASP55 is not yet available to directly test this effect. Nevertheless, it has been demonstrated that ERK and JNK are both activated downstream of PKC stimulation by phorbol 12-myristate 13-acetate (PMA) (Sriraman et al., 2008), consistent with their shared dependence on PKD. Protein kinase D2 may therefore act as a common upstream regulator of two divergent signaling branches: one operating through RAF1–MEK1 to control GRASP55, and another through JNK2 to regulate ptrevalently GRASP65. In support of this model, treatment with the MEK inhibitors UO126 and PD98059 inhibit GRASP65 phosphorylation with reduced efficiency compared to JNK inhibition. However, the MEK inhibitors are expected to inhibit GRASP55 phosphorylation during G2, pointing to a distinct, and separately regulated, signaling pathway governing GRASP65. This is now better explained in the text (lines 345 -350).

Reviewer 2 Report

Comments and Suggestions for Authors

This study focuses on the role of "PKD2" in regulating GRASP65 phosphorylation and Golgi unlinking during G2/M transition.

major concerns:

1. The title uses "PKD2" which is kind of confusing. In pubmed search, the result of "PKD2" is for polycystic kidney disease mainly. On Uniprot search, it is difficult to make sure which protein is PKD2. Is PKD2 for this study serine/threonine protein kinase 2?Q9B2L6? PRKD2 gene? 878 amino acids? I would suggest to use full name on the title.
2. Please show some WB data to support phosphorylation of GRASP65 by PKD2. also include drug-effect on WB of PKD level as control.
3. Is S274 the only phosphorylation site for GRASP65?
4. How about T216 and S373 sites on GRAPS65?
5. Would any downstream targets of PKD2 influence GRASP65 phosphorylation?
6. Please add n=? on Figures 2A, 2C, 3A, 3C, 4A, 4B, 5B, 5D, 5E.
7.  

Author Response

We would like to thank  the Reviewer for the time dedicated to evaluating our manuscript and for the constructive comments. We believe that addressing the Reviewers' concerns has improved the quality of the work. We have carefully addressed all the issues raised, and the changes made to the manuscript are highlighted in the revised version, which now contains an additional panel in Supplementary Figure 2C. Moreover, we have reduced the number of self-citations in the references to 6. 

R2 This study focuses on the role of “PKD2” in regulating GRASP65 phosphorylation and Golgi unlinking during G2/M transition.

Major concerns:

1. The title uses “PKD2” which is kind of confusing. In pubmed search, the result of “PKD2” is for polycystic kidney disease mainly. On Uniprot search, it is difficult to make sure which protein is PKD2. Is PKD2 for this study serine/threonine protein kinase 2? Q9B2L6? PRKD2 gene? 878 amino acids? I would suggest to use full name on the title.

We thank the Reviewer for this helpful suggestion. As correctly noted, “PKD2” is widely associated with polycystic kidney disease protein 2. We have therefore revised the title to use the full name “protein kinase D2”, encoded by the PRKD2 gene, which specifies the serine/threonine kinase studied here.

 

2. Please show some WB data to support phosphorylation of GRASP65 by PKD2. Also include drug-effect on WB of PKD level as control.

Regarding the first point, the sequence surrounding Serine 274 does not match the consensus sequence for the PKCμ/PKD family, LxRxxS/T (Nishikawa et al., J Biol Chem. 1997; 272(2)), arguing against direct phosphorylation of this site by PKD (see lanes 585-590. Moreover, unfortunately our antibody dos does not work by western blotting.

In the Figure Supplementary 2 C we now include a western blot in which cells are treated with CRT0066101 for 1 hour. As it can be observed, the PKD2 levels are not affected by the drug. (see text lines 297-8)

 

 

3. Is S274 the only phosphorylation site for GRASP65?
4. How about T216 and S373 sites on GRASP65?

GRASP65 is phosphorylated at multiple residues during G2 and mitosis. The available evidence refers mainly to the rat sequence, where the residues known to be phosphorylated include: Ser216/217, Thr220/Thr224, Ser277 (272 in human), and Ser376, all targeted by CDK1 and/or PLK1 (Tang et al. 2012, Biol. Open 1:1204–14). Each phosphorylation event contributes to distinct steps of Golgi disassembly. Note that T216 and S373 are the likely human counterparts of rat T220 and S376, respectively. Among the various residues, Ser274 (Ser277 in rat) stands out as the most relevant readout for G2-specific Golgi unlinking for several reasons. First, it is the earliest mitotically phosphorylated residue, appearing in late G2, whereas Thr220/Thr224 is not modified until prophase. Second, in G2 Ser274 is phosphorylated exclusively by JNK2, making it a specific, non-redundant readout for JNK2 activity at this stage, unlike its phosphorylation in interphase (by ERK) or in mitosis (by CDK1). Third, Sr274 functional relevance has been directly demonstrated: a membrane-permeant peptide encompassing this site, but not its Ser274A mutant counterpart, delays mitotic entry upon administration (Cervigni et al., Methods, 2025). Fourth, Ser274 lies directly downstream of the PKD2→JNK2 axis we are characterizing, making it the obligate molecular node linking PKD2 activity to Golgi unlinking and the G2/M transition. Fifth, phosphorylation of Ser373 (Ser376 in rat) by CDK1 contributes to reducing Golgi cisternae stacking and to mitotic stack disassembly, but this occurs later in the cell cycle and is therefore not specific to the G2 window. At the end of mitosis, GRASP65 is dephosphorylated as the Golgi reassembles in the daughter cells. This is now better explained in the text (lines 97-104).

 

5. Would any downstream targets of PKD2 influence GRASP6 phosphorylation? PKD is an established upstream regulator of the RAF1–MEK1 pathway (Kienzle et al., 2013), suggesting that this leads to GRASP55 phosphorylation during the G2 phase. Moreover, Feinstein & Linstedt (2007-2008) showed that ERK can phosphorylate GRASP55 to induce Golgi fragmentation. Thus, as ERK is the downstream target of MEK1, likely GRASP55 phosphorylation is also controlled by PKD, but a specific antibody against phosphorylated GRASP55 is not yet available to directly test this effect. Nevertheless, it has been demonstrated that ERK and JNK are both activated downstream of PKC stimulation by phorbol 12-myristate 13-acetate (PMA) (Sriraman et al., 2008), consistent with their shared dependence on PKD. Protein kinase D2 may therefore act as a common upstream regulator of two divergent signaling branches, one operating through RAF1–MEK1 to control GRASP55, and another through JNK2 to regulate GRASP65. In support of this model, treatment with the MEK inhibitors UO126 and PD98059 showed a reduced efficacy in reducing GRASP65 phosphorylation compared to JNK inhibition, pointing to a distinct, separately regulated signaling pathway governing GRASP65. This is now better explained in the text (lines 345 -350). Moreover, treatment with the MEK1/2 inhibitors UO126 and PD98059 had only a minor effect on GRASP65 phosphorylation (Figure 3C), indicating that distinct downstream effectors mediate GRASP65 regulation through a MEK-independent branch of the pathway.

 

6. Please add n = ? on Figures 2A, 2C, 3A,3C, 4A, 4B, 5B, 5D and

We have added the cell counts (n) for all samples in the indicated figures.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The study demonstrates that PKD2 is a key regulator of the JNK2-GRASP65 signaling axis controlling Golgi disassembly at the G2/M transition.

The study highlights the importance of the phosphorylation og GRASP65 in G2 phase.

1. Panels A and C in Figures 2 and 3 appear small. Consider making them larger and indicating n numbers.
2. On line 379, the meaning of 'PKD stimulation' is unclear. A clearer explanation of whether the stimulation is direct or indirect, or consider using activation.
3. Phosphorylation of GRASP65 in Figure 3 may be discussed in detail. SP600125 can be explained in the Figure legend.    

Author Response

We would like to thank the Reviewers for the time dedicated to evaluating our manuscript and for the constructive comments. We believe that addressing the Reviewers' concerns has improved the quality of the work. We have carefully addressed all the issues raised, and the changes made to the manuscript are highlighted in the revised version, which now contains an additional panel in Supplementary Figure 2C. Moreover, we have reduced the number of self-citations in the references to 6. 

R3 The study demonstrates that PKD2 is a key regulator of the Jnk2-GRASP65 signaling axis controlling Golgi disassembly at the G2/M  transition.

The study highlights the importance of the phosphorylation of GRASP65 in G2 phase.

1. Panels a and C in Figures 2 and 3 appear small. Consider making them larger and indicating n numbers.

The indicated panels have been enlarged and cell counts (n) have been added for each experimental condition.

 

2. On line 379, the meaning of “PKD stimulation” is unclear. A clearer explanation of whether the stimulation is direct or indirect, or consider using activation.

We thank the Reviewer for this observation. The title has been corrected to read “PKD activation” (lane 396).

 

3. Phosphorylation of GRASP65 in Figure 3 may be discussed in detail. SP600125 can be explained in the figure legend.

We have expanded the discussion of Figure 3 to indicate that treatment with SP600125, a selective JNK inhibitor, reduces the P-GRASP65 signal which is now described in the figure legend (see lane 445 and 447).

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

no more comments