The influence of inositol phosphates and phosphoinositides on the ␣ isoform of the RAC-protein kinase B (RAC/PKB) was studied using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain. Binding of inositol phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence. Inositol phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 M and 0.5 M, respectively. Similar values were obtained using RAC/PKB expressed and purified from baculovirus-infected Sf9 cells in the fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate on the activity of RAC/PKB purified from transfected COS-1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate was found to inhibit the RAC/ PKB kinase activity with half-maximal inhibition at 2.5 M. In contrast, phosphatidylinositol 3,4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 M). A mutant RAC/PKB protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate that the PH domain of RAC/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of RAC/ PKB activity. The data also provide further evidence for a link between phosphatidylinositide 3-kinase and RAC/ PKB regulation. The stimulation of receptor tyrosine kinases (RTK)1 by agonists leads to immediate activation of intracellular signal transduction pathways. The assembly of multiprotein complexes at the plasma membrane is one important feature of RTK signal transduction mechanisms (reviewed in Refs. 1 and 2). Numerous studies suggest that the activation of phosphatidylinositide 3-kinase (PI3-K) by growth factors is also involved (3, 4), leading to the accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ); these metabolites are assumed to act as second messengers (5). Recently RAC-protein kinase B (RAC/PKB) has emerged as a key player in the PI3-K-stimulated signaling pathway, based on the inhibition of its activation by wortmannin (6 -11).RAC/PKB is a subfamily of the second messenger-regulated serine/threonine kinases (12). Three isoforms (␣, ␤, ␥) have been identified, each consisting of an amino-terminal pleckstrin homology (PH) domain, a central kinase domain, and a serine/threonine-rich carboxyl-terminal region (13-17). Various stimuli, such as insulin, PDGF, epidermal growth factor, basic fibroblast growth factor, serum (6 -10), and protein phosphatase inhibitors (9), lead to activation of RAC/PKB kinase activity. The activation is promoted by signals emanating from RTK-regulated PI3-K and is accompanied by an increase in serine/threonine phosphorylation of RAC/PKB (6, 9). The phosphorylation of two sites has been ...

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The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKBalpha termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKBalpha at the plasma membrane. Binding of CTMP reduces the activity of PKBalpha by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt-transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

Src family kinases such as Lyn are important signaling intermediaries, relaying and modulating different inputs to regulate various outputs, such as proliferation, differentiation, apoptosis, migration and metabolism. Intriguingly, Lyn can mediate both positive and negative signaling processes within the same or different cellular contexts. This duality is exemplified by the B-cell defect in Lyn−/− mice in which Lyn is essential for negative regulation of the B-cell receptor; conversely, B-cells expressing a dominant active mutant of Lyn (Lynup/up) have elevated activities of positive regulators of the B-cell receptor due to this hyperactive kinase. Lyn has well-established functions in most haematopoietic cells, viz. progenitors via influencing c-kit signaling, through to mature cell receptor/integrin signaling, e.g. erythrocytes, platelets, mast cells and macrophages. Consequently, there is an important role for this kinase in regulating hematopoietic abnormalities. Lyn is an important regulator of autoimmune diseases such as asthma and psoriasis, due to its profound ability to influence immune cell signaling. Lyn has also been found to be important for maintaining the leukemic phenotype of many different liquid cancers including acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML) and B-cell lymphocytic leukaemia (BCLL). Lyn is also expressed in some solid tumors and here too it is establishing itself as a potential therapeutic target for prostate, glioblastoma, colon and more aggressive subtypes of breast cancer.Lay AbstractTo relay information, a cell uses enzymes that put molecular markers on specific proteins so they interact with other proteins or move to specific parts of the cell to have particular functions. A protein called Lyn is one of these enzymes that regulate information transfer within cells to modulate cell growth, survival and movement. Depending on which type of cell and the source of the information input, Lyn can positively or negatively regulate the information output. This ability of Lyn to be able to both turn on and turn off the relay of information inside cells makes it difficult to fully understand its precise function in each specific circumstance. Lyn has important functions for cells involved in blood development, including different while blood cells as well as red blood cells, and in particular for the immune cells that produce antibodies (B-cells), as exemplified by the major B-cell abnormalities that mice with mutations in the Lyn gene display. Certain types of leukaemia and lymphoma appear to have too much Lyn activity that in part causes the characteristics of these diseases, suggesting it may be a good target to develop new anti-leukaemia drugs. Furthermore, some specific types, and even specific subtypes, of solid cancers, e.g. prostate, brain and breast cancer can also have abnormal regulation of Lyn. Consequently, targeting this protein in these cancers could also prove to be beneficial.