Pull-down of calmodulin-binding proteins.
ABSTRACT Calcium (Ca(2+)) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca(2+) signaling is mediated through the calcium-binding protein known as calmodulin (CaM). CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca(2+) (Ca(2+)-CaM) as opposed to its Ca(2+)-free state (ApoCaM). While most target proteins bind Ca(2+)-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin, neurogranin (Ng), and certain myosins. These proteins have been shown to play important roles in presynaptic function, postsynaptic function, and muscle contraction, respectively. Their ability to bind and release CaM in the absence or presence of Ca(2+) is pivotal in their function. In contrast, many proteins only bind Ca(2+)-CaM and require this binding for their activation. Examples include myosin light chain kinase, Ca(2+)/CaM-dependent kinases (CaMKs) and phosphatases (e.g. calcineurin), and spectrin kinase, which have a variety of direct and downstream effects. The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca(2+)-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca(2+)-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM. Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca(2+), using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca(2+) on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca(2+)/CaM signaling and proteins that interact with CaM.
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ABSTRACT: Calcium entry and the subsequent activation of CaMKII trigger synaptic plasticity in many brain regions. The induction of long-term potentiation (LTP) in the CA1 region of the hippocampus requires a relatively high amount of calcium-calmodulin. This requirement is usually explained, based on in vitro and theoretical studies, by the low affinity of CaMKII for calmodulin. An untested hypothesis, however, is that calmodulin is not randomly distributed within the spine and its targeting within the spine regulates LTP. We have previously shown that overexpression of neurogranin enhances synaptic strength in a calmodulin-dependent manner. Here, using post-embedding immunogold labeling, we show that calmodulin is not randomly distributed, but spatially organized in the spine. Moreover, neurogranin regulates calmodulin distribution such that its overexpression concentrates calmodulin closer to the plasma membrane, where a high level of CaMKII immunogold labeling is also found. Interestingly, the targeting of calmodulin by neurogranin results in lowering the threshold for LTP induction. These findings highlight the significance of calmodulin targeting within the spine in synaptic plasticity.PLoS ONE 07/2012; 7(7):e41275. DOI:10.1371/journal.pone.0041275 · 3.53 Impact Factor
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ABSTRACT: Purkinje cell protein 4-like 1 (Pcp4l1) is a small neuronal IQ motif protein closely related to the calmodulin-binding protein Pcp4/PEP-19. PEP-19 interacts with calmodulin via its IQ motif to inhibit calmodulin-dependent enzymes and we hypothesized Pcp4l1 would have similar properties. Surprisingly, full-length Pcp4l1 does not interact with calmodulin in yeast two-hybrid or pulldown experiments yet a synthetic peptide constituting only the IQ motif of Pcp4l1 binds calmodulin and inhibits calmodulin-dependent kinase II. A nine-residue glutamic acid-rich sequence in Pcp4l1 confers these unexpected properties. This element lies outside the IQ motif and its deletion or exchange with the homologous region of PEP-19 restores calmodulin binding. Conversion of a single isoleucine (Ile36) within this motif to phenylalanine, the residue present in PEP-19, imparts calmodulin binding onto Pcp4l1. Moreover, only aromatic amino acid substitutions at position 36 in Pcp4l1 allow binding. Thus, despite their sequence similarities PEP-19 and Pcp4l1 have distinct properties with the latter harboring an element that can functionally suppress an IQ motif. We speculate Pcp4l1 may be a latent calmodulin inhibitor regulated by post-translational modification and/or co-factor interactions.Journal of Neurochemistry 03/2012; 121(6):843-51. DOI:10.1111/j.1471-4159.2012.07745.x · 4.24 Impact Factor