Identification of Targets for Calcium Signaling through the Copine Family of Proteins

Abstract

We provide evidence that copines, members of a ubiquitous family of calcium-dependent, membrane-binding proteins, may represent a universal transduction pathway for calcium signaling because we find copines are capable of interacting with a wide variety of "target" proteins including MEK1, protein phosphatase 5, and the CDC42-regulated kinase, that are themselves components of intracellular signaling pathways. The copine target proteins were identified by yeast two-hybrid screening and the interactions were verified in vitro using purified proteins. In the majority of cases the copine binds to a domain of the target protein that is predicted to form a characteristic coiled-coil. A consensus sequence for the coiled-coil copine-binding site was derived and found to have predictive value for identifying new copine targets. We also show that interaction with copines may result in recruitment of target proteins to membrane surfaces and regulation of the enzymatic activities of target proteins.

Copine-I represses NF-κB transcription by endoproteolysis of p65

“…The ability of the C2D to inhibit p65 transcriptional activity was unexpected (Figure 4c). C2Ds may function as dimerization domains in the presence of calcium (Tomsig et al, 2003). This published result supports our observation that copine-I functions as a dimer where the C2D recruits endogenous copine proteins.…”

“…This may reflect a more general function for the copine family of proteins in recruiting proteins bound to their ADs to membranes or to protein complexes (Tomsig et al, 2003). Immunoprecipitated copine-I, and recombinant GST-copine-I alone or mixed with cell extracts, were both unsuccessful in stimulating proteolysis of [ 35 S]-labeled p65 in vitro (data not shown).…”

“…However, we did not examine whether copine-I interacted directly with IkB family members. Copine-I was shown to preferentially bind proteins containing coiled-coil and ankyrin-repeat domains (Tomsig et al, 2003). In Caenorhabditis elegans, copine is require for spermatogenesis, and physically interacts with IkB-1, the ortholog of human atypical IkB molecule, Bcl-3 (Li et al, 2004).…”

“…Although copine-I was shown to bind several intracellular proteins with diverse biological functions (Tomsig et al, 2003), the role of copine-I in regulating biological processes is not well understood. The presence of cis-kB-binding elements in the promoter region for copine-I provided the initial rationale for investigating whether copine modulated NF-kB transcription in response to tumor necrosis factor-a (TNFa) (Tomsig et al, 2004).…”

Copine-III interacts with ErbB2 and promotes tumor cell migration

“…Copine-III is a Ca 2 þ -dependent, membrane-binding protein Copine-III belongs to a family of proteins, which have been shown to be Ca 2 þ -dependent, phospholipid binders (Creutz et al, 1998;Tomsig et al, 2003). Copine-III is ubiquitously expressed in human tissues (HUGE database); however, essentially nothing is known about its role in normal or cancer tissue.…”