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Referee comments: Referee 2

Posted by PLOS_ONE_Group on 18 Feb 2008 at 18:16 GMT

Referee 2's review:

The work of guest and colleagues reports a new role for CAMKKalpha in the monocytic response to PMA.

One of the major findings of this paper is that CAMKK is translocated to the nucleus in response to PMA stimulation. I'm concerned about the purity of the nuclear prep. In my experience, nuclei prepared in the manner described herein often co-pellet with cell membrane. The ER is often contiguous with the nuclear envelope and this is a concern. I would suggest some Western blots with markers of plasma or intracellular membranes to demonstrate that the "nuclear" fractions are not highly contaminated with membrane proteins. At the same time, Westerns for known nuclear proteins would also enhance the figures. Alternatively or in addition, immuno-staining of fixed cells could also serve this purpose.

There is an axiom that the specificity of a pharmacologic inhibitor is inversely related to the length of time it has studied. It would be helpful for future experiments to generate mutant alleles of CAMKK that are not activated in response to PMA. Taking a molecular genetic approach in addition to the pharmacologic inhibition would certainly add to the specificity and sophistication of this work.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.

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