The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.

We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM).

Collapsin response mediator proteins (CRMPs) are a family of neuron-enriched proteins that regulate neurite outgrowth and growth cone dynamics. Here, we show that Cdk5 phosphorylates CRMP1, CRMP2, and CRMP4, priming for subsequent phosphorylation by GSK3 in vitro. In contrast, DYRK2 phosphorylates and primes CRMP4 only. The Cdk5 and DYRK2 inhibitor purvalanol decreases the phosphorylation of CRMP proteins in neurons, whereas CRMP1 and CRMP2, but not CRMP4, phosphorylation is decreased in Cdk5 ؊/؊ cortices. Stimulation of neuroblastoma cells with IGF1 or TPA decreases GSK3 activity concomitantly with CRMP2 and CRMP4 phosphorylation. Conversely, increased GSK3 activity is not sufficient to increase CRMP phosphorylation. However, the growth cone collapse-inducing protein Sema3A increases Cdk5 activity and promotes phosphorylation of CRMP2 (but not CRMP4). Therefore, inhibition of GSK3 alters phosphorylation of all CRMP isoforms; however, individual isoforms can be differentially regulated by their respective priming kinase. This is the first GSK3 substrate found to be regulated in this manner and may explain the hyperphosphorylation of CRMP2 observed in Alzheimer's disease.Glycogen synthase kinase 3 (GSK3) 4 is an evolutionarily conserved and ubiquitously expressed Ser/Thr kinase that is expressed as two closely related isoforms in mammals, GSK3␣ (51 kDa) and GSK3␤ (47 kDa) (1). GSK3 is unusual when compared with other protein kinases as it is constitutively active in cells and phosphorylation of most substrates must be preceded by phosphorylation of a nearby residue by another kinase. This process is known as priming and occurs at Ser/Thr residues located 4 or 5 residues C-terminal to the site phosphorylated by GSK3 (2, 3). GSK3 activity is inhibited by phosphorylation of an N-terminal serine residue (Ser 21 on GSK3␣ and Ser 9 on GSK3␤), which is catalyzed by members of the AGC family of protein kinases upon stimulation by growth factors (4, 5). Alternatively, GSK3 activity may be inhibited by protein-protein interactions following activation of the Wnt signaling pathway (6, 7). It is also possible that regulation of priming kinases could indirectly regulate phosphorylation of substrates by GSK3, although this has yet to be proven.We have recently discovered new brain-specific substrates for GSK3, namely collapsin response mediator protein (CRMP) 2 and 4 (3). These isoforms are members of a family of five CRMP proteins (CRMP1-5) that are expressed almost ubiquitously throughout the central nervous system (8, 9). CRMP2 is the best studied isoform of the family. Mammalian CRMP2 binds to tubulin heterodimers to promote microtubule formation and co-localizes with microtubules inside cells (10). Overexpression of CRMP2 in hippocampal neurons promotes increased axon elongation (3, 10, 11). However, mutation of the GSK3 phosphorylation sites on CRMP2 to non-phosphorylatable alanine residues alters CRMP2-induced axon elongation (3, 12). Other functions attributed to CRMP2 include regulation of cell surface receptor intern...

We have identified Rab5 as a critical cytosolic component required for clathrin-coated pit function. Given the well-established role of Rab5 in the fusion of endocytic vesicles with endosomes, our results suggest that recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.

Protein kinase activity results in the incorporation of radiolabeled phosphate from [gamma-32P]ATP into a peptide or protein substrate. The measurement of the amount of radioactivity incorporated into a substrate as a function of time and enzyme concentration allows enzyme activity to be quantified. The activity is expressed as a 'unit', where 1 unit corresponds to the amount of protein kinase that catalyzes the incorporation of 1 nanomole of phosphate into the standard substrate in 1 minute. Specific activity is defined as units of activity per milligram protein. The assay format described here is quick, simple, inexpensive, sensitive and accurate, provides a direct measurement of activity and remains the 'gold standard' for the quantification of protein kinase activity. Up to 40 samples can be assayed manually at one time, and the assay takes one person less than 1 hour to complete.