Rabbit Anti-Human LIMK2 monoclonal antibody, clone U.867.2

BACKGROUND: Cognitive dysfunction in schizophrenia is associated with a lower density of dendritic spines on deep layer 3 pyramidal cells in the dorsolateral prefrontal cortex (DLPFC). These alterations appear to reflect dysregulation of the actin cytoskeleton required for spine formation and maintenance. Consistent with this idea, altered expression of genes in the cell division cycle 42 (CDC42)-CDC42 effector protein (CDC42EP) signaling pathway, a key organizer of the actin cytoskeleton, was previously reported in DLPFC gray matter from subjects with schizophrenia. We examined the integrity of the CDC42-p21-activated serine/threonine protein kinases (PAK)-LIM domain-containing serine/threonine protein kinases (LIMK) signaling pathway in schizophrenia in a layer-specific and cell type-specific fashion in DLPFC deep layer 3. METHODS: Using laser microdissection, samples of DLPFC deep layer 3 were collected from 56 matched pairs of subjects with schizophrenia and comparison subjects, and levels of CDC42-PAK-LIMK pathway messenger RNAs were measured by quantitative polymerase chain reaction. These same transcripts also were quantified by microarray in samples of individually microdissected deep layer 3 pyramidal cells from a subset of the same subjects and from monkeys exposed to antipsychotics. RESULTS: Relative to comparison subjects, CDC42EP4, LIMK1, LIMK2, ARHGDIA, and PAK3 messenger RNA levels were significantly upregulated in subjects with schizophrenia in laminar and cellular samples. In contrast, CDC42 and PAK1 messenger RNA levels were significantly downregulated specifically in deep layer 3 pyramidal cells. These differences were not attributable to psychotropic medications or other comorbid factors. CONCLUSIONS: Findings from the present and prior studies converge on synergistic alterations in CDC42 signaling pathway that could destabilize actin dynamics and produce spine deficits preferentially in deep layer 3 pyramidal cells in schizophrenia.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death. In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by y[P-32] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.