Swiprosin-1 modulates actin dynamics by regulating the F-actin accessibility to cofilin

Membrane protrusions, like lamellipodia, and cell movement are dependent on actin dynamics, which are regulated by a variety of actin-binding proteins acting cooperatively to reorganize actin filaments. Here, we provide evidence that Swiprosin-1, a newly identified actin-binding protein, modulates lamellipodial dynamics by regulating the accessibility of F-actin to cofilin. Overexpression of Swiprosin-1 increased lamellipodia formation in B16F10 melanoma cells, whereas knockdown of Swiprosin-1 inhibited EGF-induced lamellipodia formation, and led to a loss of actin stress fibers at the leading edges of cells but not in the cell cortex. Swiprosin-1 strongly facilitated the formation of entangled or clustered F-actin, which remodeled the structural organization of actin filaments making them inaccessible to cofilin. EGF-induced phosphorylation of Swiprosin-1 at Ser183, a phosphorylation site newly identified using mass spectrometry, effectively inhibited clustering of actin filaments and permitted cofilin access to F-actin, resulting in actin depolymerization. Cells overexpressing a Swiprosin-1 phosphorylation-mimicking mutant or a phosphorylation-deficient mutant exhibited irregular membrane dynamics during the protrusion and retraction cycles of lamellipodia. Taken together, these findings suggest that dynamic exchange of Swiprosin-1 phosphorylation and dephosphorylation is a novel mechanism that regulates actin dynamics by modulating the pattern of cofilin activity at the leading edges of cells.     

Ikaros negatively regulates inducible nitric oxide synthase expression in macrophages: Involvement of Ikaros phosphorylation by casein kinase 2

Ikaros is known as a critical regulator of lymphocyte development. We examined the regulatory role of Ikaros in LPS/IFN-gamma-induced inducible nitric oxide synthase (iNOS) expression by macrophages. Our results showed that IK6 (Ikaros dominant negative isoform) induction increases the iNOS expression. Ikaros DNA binding activity on the iNOS promoter was decreased, and a mutation of the Ikaros-binding site on the iNOS promoter resulted in an increase in LPS/IFN-gamma-induced iNOS expression. LPS/IFN-gamma increased the histone (H3) acetylation on the Ikaros DNA binding site. These results suggest that Ikaros acts as a negative regulator on iNOS expression. Treatment with a casein kinase 2 (CK2) inhibitor reversed LPS/IFN-gamma-induced decrease in Ikaros DNA binding activity. Moreover, overexpression of kinase-inactive CK2 decreased iNOS expression and a significant amount of CK2alpha1 translocated into the nucleus in LPS/IFN-gamma-treated cells. Overall, these data indicate that LPS/IFN-gamma decreases the Ikaros DNA binding activity via the CK2 pathway, resulting in an increase of iNOS expression.     

Inactivation of human glutamate dehydrogenase by aluminum

Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a k_inact of 2.7 min^-1 and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP⁺, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 M. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD⁺-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in helices and sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.Received 25 July 2003; received after revision 27 August 2003; accepted 15 September 2003     

SPIN90 dephosphorylation is required for cofilin-mediated actin depolymerization in NMDA-stimulated hippocampal neurons

Actin plays a fundamental role in the regulation of spine morphology (both shrinkage and enlargement) upon synaptic activation. In particular, actin depolymerization is crucial for the spine shrinkage in NMDAR-mediated synaptic depression. Here, we define the role of SPIN90 phosphorylation/dephosphorylation in regulating actin depolymerization via modulation of cofilin activity. When neurons were treated with NMDA, SPIN90 was dephosphorylated by STEP61 (striatal-enriched protein tyrosine phosphatase) and translocated from the spines to the dendritic shafts. In addition, phosphorylated SPIN90 bound cofilin and then inhibited cofilin activity, suggesting that SPIN90 dephosphorylation is a prerequisite step for releasing cofilin so that cofilin can adequately sever actin filaments into monomeric form. We found that SPIN90 YE, a phosphomimetic mutant, remained in the spines after NMDAR activation where it bound cofilin, thereby effectively preventing actin depolymerization. This led to inhibition of the activity-dependent redistribution of cortactin and drebrin A, as well as of the morphological changes in the spines that underlie synaptic plasticity. These findings indicate that NMDA-induced SPIN90 dephosphorylation and translocation initiates cofilin-mediated actin dynamics and spine shrinkage within dendritic spines, thereby modulating synaptic activity.