A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells.

Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin(-/-) mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division

Anchored phosphatases modulate glucose homeostasis.

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic β-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-β-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in β-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization

(GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. Conclusion: 14-3-3 proteins can potentially engage around 0.6 % of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been 1Samuel Lunenfeld Research Institute validated by the broad inhibition of 14-3-3 phosphoryla-Mount Sinai Hospital tion-dependent binding in vivo and by the specific analy-600 University Avenue sis of AKAP-Lbc, a RhoGEF that is controlled by it

Gravin, an autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein

AbstractBackground: Subcellular targeting of protein kinases and phosphatases provides a mechanism for co-localizing these enzymes with their preferred substrates. A recently identified mammalian scaffold protein, AKAP79, controls the location of two broad-specificity kinases and a phosphatase.Results: We have identified and characterized another mammalian scaffold protein which coordinates the location of protein kinase A and protein kinase C. We isolated a cDNA encoding a 250 kDa A-skinase anchoring protein (AKAP) called gravin, which was originally identified as a cytoplasmic antigen recognized by myasthenia gravis sera. Sequence homology to proteins that are known to bind protein kinase C suggests that gravin also binds this kinase. Studies of binding in vitro show that residues 1526–1780 of gravin bind the regulatory subunit (RII) of protein kinase A with high affinity, and residues 265–556 bind protein kinase C. Gravin expression in human erythroleukemia cells can be induced with phorbol ester, resulting in the detection of a 250 kDa RII-  and PKC-binding protein. Immunolocalization experiments show that gravin is concentrated at the cell periphery and is enriched in filopodia. Gravin staining is coincident with an AKAP detected by an in situ RII-overlay assay, and a PKA–gravin complex can be isolated from human erythroleukemia cells.Conclusions: We present biochemical evidence that gravin forms part of a signaling scaffold, and propose that protein kinases A and C may participate in the coordination of signal transduction events in the filopodia of human erythroleukemia cells

Plugging PKA into ERK scaffolds

Cancers often arise in part through derangements in protein kinase signaling. A striking example of this is the finding that approximately 30% of human tumors have mutations in Ras or B-Raf, leading to aberrant ERK kinase activation. Kinase signaling networks are often organized by scaffolding and anchoring proteins that help shape the dynamics of signal processing. AKAP-Lbc associates with the ERK scaffold protein KSR-1 to organize a growth factor and cAMP responsive signaling network. AKAP-Lbc also directs PKA phosphorylation of KSR-1 on a critical residue to ensure maximal signaling efficiency