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Metrics details. Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions.

These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity.

Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility. Kinases are conserved during evolution. Orthologs with 'kinase domains' so-called protein kinase-like folds; PKL are found in all three domains of life, [ 1 ].

Interestingly, comparing the 'kinomes' from nematodes, insects, and vertebrates a surprising number of kinases are shared. The human genome contains putative protein and lipid kinases. Based on sequence similarities they can be divided into 9 groups of conventional kinases, which feature a typical kinase domain sequence, and 8 small groups of unconventional kinases, which lack typical kinase domain sequences but reportedly possess biochemical kinase activity [ 2 , 3 ].

Almost half of the human kinases can be mapped to known disease loci, cancer amplicons, and mutations or their deregulation can be directly correlated to human disease. Therefore, it comes as no surprise that kinases are intensively studied, and kinase inhibitors have now a firm place in the pharmaceutical armoury.

The importance of protein phosphorylation is underlined by a Nobel Prize in Physiology or Medicine awarded to Edmond H. Fischer and Edwin G. Krebs in "for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism". Their key discovery about 55 years ago was that the conversion of the inactive enzyme phosphorylase b to the active phosphorylase a is caused by phosphorylation, and that the conversion factor is a protein kinase, phosphorylase kinase [ 4 , 5 ].

That breakthrough has established a firm role for protein kinases in the regulation of diverse fundamental cellular processes and spawned an immensely fruitful field of kinase research. At the same time this success, however, has blinkered us to solely concentrate on the catalytic activities of kinases neglecting other functions of these proteins, which do not require the phosphotransferase activity.

Early examples of non-catalytic functions of protein kinases were discovered in yeast. In Posas and Saito showed that the yeast Pbs2p protein can serve both as a scaffolding protein and a protein kinase [ 6 ]. The adaptation of the yeast S. In the other pathway Pbs2p functions both as a kinase and a scaffold by assembling a complex of Sho1p, Ste11p, and Hog1p proteins and at the same time providing the kinase link between Ste11p and Hog1p Figure 1A.

In another example from budding yeast, Madhani and colleagues showed in that the MAPK Kss1 has important non-catalytic functions [ 7 ]. Non-phosphorylated Kss1 inhibits filamentation and haploid invasion through the kinase independent inhibition of the SteTec1 transcription factor complex. This second function of Kss1 requires its kinase activity, which acts to stimulate the SteTec1 complex Figure 1B.

Examples of catalytic-independent functions in yeast. A The dual role of PBS2 as scaffold and kinase in yeast osmo-sensing pathways. B Antagonistic functions of the kinase Kss1 in filamentation and invasion. Although these discoveries are not only early but still some of the clearest examples for a dual function of protein kinases, this new conceptual view did not gain traction until the last few years where a flurry of other examples began to emerge and are now receiving sharply increasing attention.

This review presents a synopsis of protein kinase functions that are independent of catalytic activity, with a special focus on kinases involved in the regulation of proliferation, apoptosis, differentiation, cell adhesion and migration. A comprehensive summary is given in Table 1. Due to space constraints we do not discuss the functions of pseudokinases, but only review recent results suggesting that in some cases their catalytic activities have developed to become highly specialized rather than being lost.

Receptor Tyrosine Kinases RTK comprise a family of about 60 cell surface receptors, which act as docking platforms for polypeptide-based growth factors, cytokines, and hormones [ 8 ]. RTKs are starting points for several signalling pathways, and hence are not only key regulators of many normal cellular processes, but also play a major role in development and progression of many malignancies [ 9 ].

Among the 60 receptors of the RTK family, several family members are involved in mechanisms where no kinase activity is required. The ERBB family of RTKs is one of the best known and most extensively studied signal transduction networks with implications ranging from cell division to cell death and motility to cellular adhesion extensively reviewed in [ 10 , 11 ]. EGFR forms homodimers as well as three functional heterodimers with the other members of the ERBB family, which stimulates its intrinsic intracellular protein-tyrosine kinase activity and results in autophosphorylation of several tyrosine residues in the C-terminal domain of EGFR [ 13 ].

These phospho-tyrosines serve as docking sites for an array of signal transducers [ 14 ], including kinases, phosphatases, transcription factors, and several adaptor proteins such as GRB2 and Shc, which are responsible for the initiation of multiple downstream signalling pathways. Interestingly, in cancer tissues the expression level of EGFR is correlated with prognosis, but not with responsiveness to EGFR inhibitor treatment [ 15 ].

This conundrum suggests that EGFR might contribute to tumor progression independently of its kinase activity. Several studies support this kinase-independent pro-survival function of the EGFR. While EGFR knockout animals die soon after birth [ 17 ], animals expressing kinase-defective EGFR are viable and display only some epithelial defects [ 18 ].

Second, several groups reported the surprising results, that a kinase-defective EGFR was capable to activate downstream signalling such as MAPK and stimulate DNA synthesis, while failing to induce the tyrosine phosphorylation of endogenous substrates in response to EGF [ 19 — 22 ].

These kinase activity independent signalling included transcriptional effects, as kinase-deficient EGFR could activate c-fos expression [ 23 ]. The mechanism may include heterodimerization with other ERBB members. ERBB2's kinase activity, but not tyrosine phosphorylation, was required for this activation. These results suggest that EGFR has catalytic-independent functions, which might be achieved by heterodimerization with other members of the ERBB receptor family.

More recent studies provide more mechanistic insights as to the nature of kinase-independent signalling, which relies on protein-protein interactions. In , Weihua et al. Interestingly, inhibition of the EGFR kinase activity did not block this association with SGLT1 or decrease basal intracellular glucose levels suggesting that no kinase activity is required for this regulation.

These results have important implications for therapeutic approaches relying on agents that inhibit the EGFR kinase activity, as the kinase independent functions of EGFR may open escape routes, which for instance maintain the viability of tumor cells even in the presence of EGFR kinase inhibitors.

In addition to non-catalytic functions regulating downstream effectors by the EGFR via direct protein interactions, they also contribute to the regulation of the localisation of the EGFR family itself.

Ligand binding induces the internalization of the receptor into endosomes, where the receptors are either targeted for ubiquitin-mediated degradation or recycled back to the plasma membrane. While this process was thought to require kinase activation, newer data suggest that rather than EGFR kinase activity, dimerization is necessary and sufficient for internalization [ 26 ].

ERBB family receptors contain nuclear localisation signals [ 27 ] enabling them to translocate to the nucleus, either as full length molecules e. However, it is unclear whether kinase activity is needed for nuclear translocation or the actual transcriptional transactivation function of the nuclear EGFR. Interestingly, a C-terminal ERBB4 fragment lacking the kinase domain is able to activate transcription by associating with the YAP2 transcription factor [ 30 ], suggesting that the transactivation function may be independent of catalytic activity.

This is not surprising given that the function of RTKs relies heavily on their abilities to assemble multi-protein signaling complexes. Although the focus has been on proteins recruited to tyrosine phosphorylation dependent docking sites, there is increasing evidence that a great number of proteins are associated with RTKs independently of ligand, and that at least some of these proteins also participate in the regulation of signaling [ 33 ].

Another recent example for kinase independent signaling is the IGFR, which could stimulate the ERK pathway despite having its kinase activity blocked by chemical IGFR inhibitors or even abolished by mutation [ 34 ]. MAPK pathways are ubiquitous signaling modules consisting of a three-tiered, and sometimes four-tiered, cascade of kinases that is activated by a small G-protein as input Figure 3A.

The name mitogen activated protein kinase is historic, but now indicates a range of pathways that respond to a variety of stimuli including mitogens, hormones, and stress signals [ 35 ]. While signaling within the cascade is largely linear, the terminal MAPK usually has a large number of substrates, whose phosphorylation kinetics and localization contribute to the generation of specific biological outputs [ 35 , 36 ].

The design of MAPK modules conveys interesting intrinsic properties, such as switchlike responses and output stabilization [ 37 , 38 ]. Kinase-independent functions of Raf kinases. D Raf-1 and A-Raf bind and inhibit the pro-apoptotic mammalian sterile like kinase MST2 thereby interfering with its dimerization, autophosphorylation, and activation. ERK features more than substrates thereby regulating many fundamental cellular functions, including proliferation, differentiation, transformation, apoptosis and metabolism [ 39 ].

From an evolutionary point of view and phylogenetic comparisons, the single Raf homologs in invertebrates lin in Caenorhabditis elegans and D-Raf in Drosophila melanogaster are much closer related to B-Raf in terms of sequence, than Raf-1 and A-Raf [ 41 , 42 ].

Furthermore, gene ablation experiments in mice showed that Raf-1 is required for survival and protects against apoptosis [ 43 , 44 ]. Taken together, these results suggested, that Raf-1 and A-Raf might possess kinase-independent functions. During the hunt for new Raf targets, new kinase-independent roles for Raf proteins besides the MEK substrate have emerged.

Over the years it emerged that Raf proteins are able to homo- and heterodimerize with each other [ 48 — 50 ]. Interestingly, these allosteric mechanisms of dimerization regulate kinase activity of the complexes. One intriguing phenomenon was that even a catalytically compromised B-Raf was capable of inducing kinase activity of Raf-1 in trans in a manner dependent on a physical interaction between B-Raf and Raf-1, suggesting that the underlying mechanism is independent of a simple transautophosphorylation route [ 46 , 51 — 53 ].

Only recently, the exact mechanism of how these dimers are regulated was discovered [ 54 ], which suggests that two Raf proteins are found in a 'side-to-side' dimer configuration. Several proteins seem to be responsible for the correct configuration, which include the scaffold KSR and proteins [ 54 ].

What has not been solved yet is the mechanism how a kinase dead Raf protein can stimulate the activity of another Raf protein in the context of a heterodimer, but an allosteric mechanism is the most plausible possibility. The observation that Raf-1 activation by heterodimerization with B-Raf seems to proceed differently from the activation used by growth factors [ 51 ], is in keeping with such an alternative mechanism of Raf activation exerted by allosteric changes. This enhancement may reflect allosteric cooperativity between Raf and KSR, and maybe other MEK kinases, when assembled into multi-protein complexes in cells.

This kind of complex formation may also play a pathophysiological role in cancer. It could explain the surprising finding that a small number of B-Raf mutations occurring in tumors have reduced kinase activity, and exert their oncogenic action by stimulating Raf-1 [ 52 ].

A therapeutically even more important observation is that Raf inhibitory drugs can activate the ERK pathway, and in clinical trials may be responsible for some adverse side effects of otherwise highly efficacious Raf inhibitors [ 60 , 61 ].

This paradoxical activation of ERK occurs in tumor cells with Ras mutations, which cooperate with Raf inhibitors to induce B-Raf-Raf-1 heterodimerization [ 62 ]. As the Raf heterodimer activates MEK at least fold stronger than B-Raf, but only requires one Raf partner to have kinase activity [ 53 ], even a slightly incomplete inhibition of Raf will promote ERK pathway activation by Raf heterodimers. This in trans regulation of a kinase domain by the regulatory domain of another kinase introduces a new concept of kinase regulation that may have important implications for signal coordination where the activation of one pathway automatically would inhibit another pathway.

The other proapoptotic kinase, which is inhibited by Raf-1, is mammalian sterile like kinase, MST2, which was identified in a proteomics screen of Rafassociated proteins [ 68 ]. MST2 is activated by dimerization and autophosphorylation of the activation loop.

Raf-1 kinase activity is not required for this regulation as kinase-dead Raf-1 mutants, or even the mutant lacking the complete kinase domain, also can inhibit MST2 activation. Interestingly, the RafMST2 interaction is induced by stress and relieved by mitogens. Upon stimulation of cells, Ras binding to Raf-1 enables Raf-1 to activate the ERK pathway and promote proliferation, but at the same time dissociates the MST2-Raf-1 complex and promotes apoptosis [ 70 ].

Coupling cell proliferation to the risk of cell death seems paradoxical at first sight, but this dual role makes perfect sense for higher organisms where the uncontrolled proliferation of cells can lead to severe diseases including cancer.

In a subsequent study, we could show that A-Raf, the third member of the Raf family, also binds to and inhibits MST2 [ 71 , 72 ]. B-Raf binds MST2 only very weakly [ 68 ]. Thus, the observed differential MST2 binding pattern inversely correlates with the kinase activity towards MEK and the evolution of the Raf family. This observation suggests that during evolution the role of Raf might have shifted from activating the ERK pathway to inhibiting the MST2 pathway.

Another kinase, which is regulated by Raf-1 independent of its catalytic activity is apoptosis signal-regulating kinase-1 ASK Figure 3C.

Raf-1 binds to ASK1 inhibiting its kinase activity and apoptosis. Although the direct mechanism of inhibition is not known yet, the pathophysiological relevance of ASK1 inhibition by Raf-1 was demonstrated in a mouse model of heart disease [ 75 ].

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The secret life of kinases: functions beyond catalysis

Owing to the increasingly globalized nature of the cyclodextrin CyD -related science and technology, development of the CyD-based pharmaceutical formulation is rapidly progressing. The pharmaceutically useful CyDs are classified into hydrophilic, hydrophobic, and ionic derivatives. This review outlines the current application of CyDs in drug delivery and pharmaceutical formulation, focusing on the following evidences. On the basis of the above-mentioned knowledge, the advantages and limitations of CyDs in the design of advanced dosage forms will be discussed. Cyclodextrins as drug carriers in Pharmaceutical Technology: The state of the art. Cyclodextrins CDs are versatile excipients with an essential role in drug delivery, as they can form non-covalently bonded inclusion complexes host-guest complexes with several drugs either in solution or in the solid state. The main purpose of this publication was to carry out a state of the art of CDs as complexing agents in drug carrier systems.

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