Dear Dr. ***:

Your manuscrlpt entitled "Title" has been reviewed by a member of the Editorial Board of the Journal of **** . with the aid of an outside referee who is well qualified to judge your studies. As you can see from the enclosed reviews, several concerns have been raised which preclude your manuscript from being accepted for publication in the Journal **** . Although one referee was positive about some aspects of the studies, both reviewers had serious concerns about the indirect nature of the experiments. The Editorial Board member, in particular, noted that neither calcium pump nor protein kinase activities were measured direetly, which makes firm conclusions about distinct regulatory effects impossible to draw. Both reviewers viewed the paper as incomplete and very inconclusive. For these reasons, I am sorry to inform you that your manuscript cannot be accepted for publication in the Journal. I hope that the enclosed comments will be helpful in your future work with this system.

Enclosed are the original figures, some under separate cover, along with the reviewep's comments. Thank you for submitting your manuscript to the Journal ***.

Sincerely

Referee Form

Date mailed to referee

MS#: ****

Author(s): Author et al_

TitIe: Title

Detailed review to be sent to the author should be included on a separate sheet in triplicate.

REMARKS:

In this manusclipt the authols examine the underlying basis for ATP evoked oscillatory Ca2+ sensitive K+ currents in megakaryocytes isolated from rat bone marrow, assumed to leflect Ca2+ oscillations in these cells. The authors have previously reported an involvement of Ca2+ ATPases and are now presenting new data which suggests the involvment of two classes of Ca2+ pumps accordiug to their sensitivity of phosphorylation-modulatfon drugs. The authors clearly show that PKC , PKA aud CaM kinase affect oscillajions. their technique is sound. However, the interpretation of their data appears limited. Why have the authors have focused solely on the Ca2+ pumps as the site of kinase action and not the IP3R. The IP3R has also been reported to be phosphorylated by each of these kinases. This is a serious problem and needs to be addressed. The previous report referred to by Author et al (JBC vol 268. 168-74), where the authors argue for the involvement of Ca2+ pumps is also not clear on this point. Although Ca2+ pumps have clearly been shown to increase Ca2+ oscillations in other systems (e.g. Sci vol. 260, p. 226-229, which the authors should site), other possible mechanisms need to be considered and discussed before they are excluded outright.

Manuscript No. ***

Date:

Authors: author et al.

Title: Title

This mauusaipt is a continuation of the authors' previous work; in which inhibitors of intracellular Ca pumps are shown to affect Ca-sensitive potassium currents in megakaryocytes. In this current paper, the effects of various protein kinase activators and inhibitors are tested.

The main conclusion of the paper-that two types of intracellular calcium pumps function in megakaryocytes-is not strongly supported by the data This is because all observations are based on indirect measurements. Calcium pump activities are not measured directly. but are inferred from the action of calcium pump inhibitors on potassium currents. Similarly, protein kinase effects are only inferred from indirect effects of added inhibitors and activators on potassium currents, and it is impossible to know if the wide array of putative protein kinase-selective drugs and peptides tested acted proximal or distal to Ca pumps or, in fact, had no direct effects on calcium pumps at all.

lh summary, I find this study to be very incomplete and unconvincing. The experiments seem to be more "polypharmacology" than biochemistry, and not typical of the type of study that is usually published in the J.