The inactivation of glycogen synthase kinase (GSK)3 has been proposed to play important roles in insulin and Wnt signalling. To define the role that inactivation of GSK3 plays, we generated homozygous knockin mice in which the protein kinase B phosphorylation sites on GSK3a (Ser21) and GSK3b (Ser9) were changed to Ala. The knockin mice were viable and were not diabetic. Using these mice we show that inactivation of GSK3b rather than GSK3a is the major route by which insulin activates muscle glycogen synthase. In contrast, we demonstrate that the activation of muscle glycogen synthase by contraction, the stimulation of muscle glucose uptake by insulin, or the activation of hepatic glycogen synthase by glucose do not require GSK3 phosphorylation on Ser21/ Ser9. GSK3 also becomes inhibited in the Wnt-signalling pathway, by a poorly defined mechanism. In GSK3a/ GSK3b homozygous knockin cells, Wnt3a induces normal inactivation of GSK3, as judged by the stabilisation of b-catenin and stimulation of Wnt-dependent transcription. These results establish the function of Ser21/Ser9 phosphorylation in several processes in which GSK3 inactivation has previously been implicated.

p38 mitogen-activated protein kinases (MAPKs) are activated primarily in response to inflammatory cytokines and cellular stress, and inhibitors which target the p38␣ and p38␤ MAPKs have shown potential for the treatment of inflammatory disease. Here we report the generation and initial characterization of a knockout of the p38␤ (MAPK11) gene. p38␤ ؊/؊ mice were viable and exhibited no apparent health problems. The expression and activation of p38␣, ERK1/2, and JNK in response to cellular stress was normal in embryonic fibroblasts from p38␤ ؊/؊ mice, as was the activation of p38-activated kinases MAPKAP-K2 and MSK1. The transcription of p38-dependent immediate-early genes was also not affected by the knockout of p38␤, suggesting that p38␣ is the predominant isoform involved in these processes. The p38␤ ؊/؊ mice also showed normal T-cell development. Lipopolysaccharide-induced cytokine production was also normal in the p38␤ ؊/؊ mice. As p38 is activated by tumor necrosis factor, the p38␤ ؊/؊ mice were crossed onto a TNF⌬ARE mouse line. These mice overexpress tumor necrosis factor, which results in development symptoms similar to rheumatoid arthritis and inflammatory bowel disease. The progression of these diseases was not however moderated by knockout of p38␤. Together these results suggest that p38␣, and not p38␤, is the major p38 isoform involved in the immune response and that it would not be necessary to retain activity against p38␤ during the development of p38 inhibitors.

PKB/Akt, S6K, SGK and RSK are mediators of responses triggered by insulin and growth factors and are activated following phosphorylation by 3-phosphoinositide-dependent protein kinase-1 (PDK1). To investigate the importance of a substrate-docking site in the kinase domain of PDK1 termed the`PIFpocket', we generated embryonic stem (ES) cells in which both copies of the PDK1 gene were altered by knock-in mutation to express a form of PDK1 retaining catalytic activity, in which the PIF-pocket site was disrupted. The knock-in ES cells were viable, mutant PDK1 was expressed at normal levels and insulin-like growth factor 1 induced normal activation of PKB and phosphorylation of the PKB substrates GSK3 and FKHR. In contrast, S6K, RSK and SGK were not activated, nor were physiological substrates of S6K and RSK phosphorylated. These experiments establish the importance of the PIF-pocket in governing the activation of S6K, RSK, SGK, but not PKB, in vivo. They also illustrate the power of knock-in technology to probe the physiological roles of docking interactions in regulating the speci®city of signal transduction pathways.

We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol 3,4,5-tris-phosphate (PtdIns(3,4,5)P3) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that PtdIns(3,4,5)P3 binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that PtdIns(3,4,5)P3 stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the PtdIns(3,4,5)P3 binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.

We explored three approaches to create tissue-specific knock-in mice by generating knock-in mice in which a substrate-docking site of the PDK1 protein kinase was ablated in Cre-expressing tissues in a way that prevented activation of one of its substrates, p70 ribosomal S6 kinase (S6K), but not another (protein kinase B (PKB)). Employing two of the approaches, termed the "heterozygous" and "minigene" methods, we generated mice in which Cre-expressing skeletal and cardiac muscle produced the mutant rather than wild type PDK1. Consistent with this, injection of these mice with insulin only induced activation of PKB but not S6K in muscle tissues. We have also demonstrated that insulin-stimulated glucose uptake proceeds normally in knock-in mice, consistent with the notion that PKB mediates this process. In contrast to conditional knock-out of PDK1 in muscle, the knock-in mice did not develop dilated cardiomyopathy, suggesting that PKB plays a key role in protecting mice from heart failure. The third knock-in strategy that was evaluated, termed the "inversion" method, did not proceed with high efficiency. We discuss the merits and disadvantages of each of the conditional knock-in approaches, along with the applications for which they may be most suited, and suggest how they could be further refined.The ability to generate targeted mutation of proteins in mice by using knock-in methodologies is a powerful approach to investigating the physiological roles of disease-causing mutations and the functional roles of domains (1-4). The knock-in approach also has significant advantages over conventional knock-out approaches that are currently more widely used to analyze the function of genes (reviewed in Ref. 5). To date most knock-in mutations have been carried out in a manner that leads to the mutant gene being present in all cells; however, this can have drawbacks, for instance premature lethality or unwanted phenotypes. Embryonic lethality in conventional knock-outs can be overcome by employing Cre/LoxP recombinase methodology, in which the exons to be deleted are flanked with 34-base pair DNA recognition sites (LoxP) for the P1 bacteriophage Cre recombinase (6). These mice are crossed to transgenic mice that express Cre in specific tissues or developmental stages. In tissues expressing Cre, efficient recombination between the LoxP sequences takes place, resulting in the excision of the intervening DNA sequence and ablation of gene expression (6).The ability to generate conditional knock-in mice using Cre/LoxP technology would be very beneficial; however, the methodology is less well characterized. In Fig. 1 we summarize three approaches that could be employed to generate tissuespecific knock-in mice. In the first strategy termed the "heterozygous" method, a mouse is generated in which one allele comprises the wild type exon flanked with LoxP sites, whereas the second allele contains the knock-in exon and is not flanked with LoxP sites. In tissues that express Cre, the wild type exon will be excised leaving only expressi...