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14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-zeta and facilitate coupling of PKC-zeta to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-zeta activity. Using recombinant 14-3-3 proteins and purified PKC-zeta in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-zeta activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-zeta by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-zeta with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-zeta. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-zeta activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-zeta, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-zeta (PKC-zeta) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-zeta-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-zeta and Raf-1 is mediated strongly by the 14-3-3beta and -theta; isotypes, but not by 14-3-3zeta. Far-Western blotting revealed that 14-3-3 binds PKC-zeta directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3beta and Raf-1. Treatment of PKC-zeta with lambda protein phosphatase also reduced its binding to 14-3-3beta in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3beta facilitated PKC-zeta binding. Together, the results suggest that 14-3-3 binds both PKC-zeta (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-zeta mutant than with wild-type PKC-zeta, supporting the idea that kinase activity leads to complex dissociation. 14-3-3beta and -θ were substrates for PKC-zeta, whereas 14-3-3zeta was not. Phosphorylation of 14-3-3beta by PKC-zeta negatively regulated their physical association. 14-3-3beta with its putative PKC-zeta phosphorylation sites mutated enhanced co-precipitation between PKC-zeta and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-zeta weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-zeta to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-zeta.

The aim of the present study was to investigate the effect of exercise duration on the amount of histological change (degeneration and cellular response), the number of affected muscle fibres, the segment over which muscle fibres were affected and the spatial distribution of affected muscle fibres in the rat soleus muscle after submaximal uphill running. Male Wistar rats ran on a treadmill (10 degrees incline, 1840 m/h) for 1.5 or 2.5 h. Three days postexercise the left and right soleus muscles were removed. Serial transverse sections were cut from proximal to distal, so that individual fibres could be followed over their entire length. Histological changes were quantified morphologically. With increasing exercise duration, an increase in the histological changes was found, as were a greater number of affected fibres and an increase in the mean length over which fibres were affected. Beside occasional minor clustering, affected fibres were distributed randomly throughout the muscle.

The innervation of the tensor tympani muscle and the stapedius muscle in the rat was studied. This was done by acetylcholinesterase in toto staining of the tympanic bullae and of muscles dissected separately, acetylcholinesterase staining of serial cross-sections of the muscles, silver impregnation of serial sections of complete tympanic bullae, serial semithin sections stained according to Laczko & Levai and electron microscopy of both muscles. The gross innervation of the muscles and the relation to other nerves in the bulla are described. It is shown that both muscles are innervated by very thin nerve fibres which form a well-organised elaborate network in the muscles, with very short branches that connect with motor endplates. Electron microscopically there are indications that the endplates in the stapedius muscle seem to enable faster activation of the muscle fibres than those of tensor tympani muscle. No morphological evidence for any sensory innervation of the muscles could be detected in the muscles themselves, in the connective tissue related to the muscles, or in the contents of the bulla tympanica. It is postulated that the afferent input of the acoustic middle ear muscle reflex is sound alone and that sensory information from the muscles themselves or from other structures in the tympanic bulla do not contribute to the reflex.

The distribution and ultrastructure of sensory nerve endings were investigated in the deep lateral elbow region of the rat. Three zones of distribution of mechanoreceptors were distinguished, each in relation to the functional architecture of the connective and muscular tissue in that area: (1) a zone with muscle spindles, Golgi tendon organs, free nerve endings and single small lamellated corpuscles ('muscle-tendon spectrum'), situated in the middle third of the supinator muscle and its superficial aponeurosis; (2) a zone with small lamellated corpuscles and free nerve endings, situated pericapsularly to the humeroradial joint capsule ('shearing spectrum'): this moderately dense, irregular connective tissue is covered by the proximal continuation of the supinator's aponeurosis, and muscle fibers insert from beneath this aponeurosis, which displays, as a part of the joint capsule, a strong collagenous tissue plate; (3) a zone with only free nerve endings within the tendon-like, most proximal part of the supinator's aponeurosis, inserting into the periosteal layer of the lateral humeral epicondyle ('endotenonial spectrum'): it is part of the joint capsule. The ultrastructure of these sensory endings is described and the distribution pattern of the mechanoreceptors observed is discussed in relation to the classification into 'muscle receptors' and 'joint receptors'.