When An Immunoblot Reagent Malfunction Leads To Important Insights: Antibody Fails To Spot Altered Kinase In Heart Cells, But Columbia Researchers Uncover Unexpected Complex Protein Regulation

Scaffolding function could point to therapeutic opportunity

BETHESDA, MD (March 14, 2006) – In the paper, “Immunoblotting PKC-delta: a cautionary note from the bench,” Columbia University researchers warn that commercial antibodies “that specifically recognize signaling proteins have become important tools in the study of signaling pathways” but the widespread use of these commercially available antibodies also can have a downside.

In a series of studies reported in the March edition of the American Journal of Physiology-Cell Physiology, the Columbia team identified “properties of certain anti-PKC-delta antibodies that affect the interpretation of immunoblotting studies.” The authors suggest that these findings related to PKC-delta may be symptomatic of a more pervasive feature of immunoblot analysis studies of phosphoproteins in general.”

The paper, “Immunoblotting PKC: a cautionary note from the bench,” appears in the March issue of the American Journal of Physiology-Cell Physiology, published by the American Physiological Society. Research was by Vitalyi O. Rybin and Susan F. Steinberg, Columbia University, New York City.

Importance of PKC-delta in heart contractility, oxidative stress and heart attack

Steinberg emphasized that the deficiencies of certain PKC-delta antibodies would have been missed had her laboratory not been engaged in very rigorous studies of PKC-delta and its regulation through phosphorylation. She said that “in trying to understand unanticipated properties of certain antibodies, which would introduce antibody artifacts if not recognized, these studies have exposed complex regulatory controls for PKC-delta through phosphorylation.”   

Steinberg noted that her laboratory has a “long-standing interest in PKC [protein kinase C] isoform functions in the heart” where they are “implicated in the regulation of cardiac contractile function, ischemic preconditioning, and structural remodeling of the heart.

 “Our recent studies have focused on PKC-delta regulation through phosphorylation events on serine/threonine and tyrosine residues in various domains of the protein.  The results suggest that tyrosine phosphorylation leads to a novel mode of PKC-delta activation during oxidative stress, since it generates an enzyme with altered co-factor requirements and substrate specificity,” she added.

During the course of studies designed to expose phosphorylation mechanisms, they discovered that “certain antibodies sold by commercial vendors don’t behave as advertised,” Steinberg said. “In particular, the BD Transduction Laboratories’ antibody is supposed to recognize the PKC-delta protein irrespective of whether this protein is ‘at rest’ or activated, that is, phosphorylated. Our study shows that this antibody doesn’t recognize the activated protein.  As a result, an unsuspecting investigator faced with these results would mistakenly conclude that there is no PKC-delta protein in tissues (or cells) that express PKC-delta in its most activated form.”

For this reason, the authors note in the AJP-Cell Physiology paper that BD Transduction’s “anti-PKC-delta antibody (which has been used widely for almost a decade)…is particularly poorly suited to comparing levels of PKC-delta expression in resting and agonist-activated samples” and that any studies using it for this purpose “deserve to be revisited using different methodologies.”

“Fortuitous findings” could point to mechanisms open to therapeutic opportunities

The paper noted that its findings “were identified somewhat fortuitously. They might have been missed had we not been pursuing studies in which we expressly focused on mechanisms controlling PKC-delta phosphorylation. Similarly the identification of discrepant properties for two different anti-PKC-delta Y-311 PSSAs also were revealed only through rather compulsive antibody characterization protocols.”  (PSSAs stands for phosphorylation site-specific antibodies.)

The recognition properties of any single PSSA, and its performance in different cellular contexts, are seldom compared by a single laboratory,” the paper and added: “However, this type of approach has become standard in the laboratories of the Alliance for Cell Signaling Technology (AfCS), where it has led to the empirical observation that a number of PSSAs, for unknown reasons, perform with the requisite sensitivity and/or specificity only in selected cellular backgrounds. This experience suggests that the problems in immunoblot analysis of PKC-delta that we have identified may not be an isolated phenomenon but may be symptomatic of a more widespread potential pitfall associated with immunoblot analysis in general.”

AfCS is a collaborative research initiative between academia and publishing “that strives to fully understand the biomedical underpinnings of signal transduction in a context-dependent manner.” The group of academic research labs is building a signaling database of molecule pages and studies. Information is available at http://www.signaling-gateway.org/.

Next steps

• “Phosphorylation apparently regulates the enzymology of PKC-delta and perhaps gives it additional as yet unidentified properties, which need to be explored,” Steinberg said.

• “It’s possible that these properties play a role in worsening cardiac ischemia or heart attack.  We are currently interested in the notion that PKC-delta acquires scaffolding function when it becomes tyrosine phosphorylated. A kinase-independent function for PKC-delta – to mislocalize interaction proteins – could open novel mechanisms that would be amenable to therapeutic interventions,” Steinberg added.

Source and funding

The paper, “Immunoblotting PKC: a cautionary note from the bench,” appears in the March 2006 issue of the American Journal of Physiology-Cell Physiology, published by the American Physiological Society. Research was by Vitalyi O. Rybin and Susan F. Steinberg, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York City.

Research was supported by grants from the United States Public Health Service-National Heart, Lung, and Blood Institute.

Editor’s note: The media may obtain a copy of Rybin and Steinberg by contacting Donna Krupa, American Physiological Society, (301) 634-7209, cell (703) 967-2751 or dkrupa@the-aps.org.

Experimental Biology 2006

Susan Steinberg is presenting “Juggling job and family:  Balancing home life and careers” as part of a symposium, “Mastering the Juggling Act: Laboratory, Life, and Leadership Roles,”
jointly sponsored by the APS Women in Physiology and ASPET Women in Pharmacology Committees, Monday April 3, Convention Center Moscone North 130 in San Francisco.

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The American Physiological Society was founded in 1887 to foster basic and applied bioscience. The Bethesda, Maryland-based society has more than 10,000 members and publishes 14 peer-reviewed journals containing almost 4,000 articles annually.

APS  provides a wide range of research, educational and career support and programming to further the contributions of physiology to understanding the mechanisms of diseased and healthy states. In May 2004, APS received the Presidential Award for Excellence in Science, Mathematics and Engineering Mentoring (PAESMEM).

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