Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylating and inactivating cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cell-cycle-dependent activity of LIMK2, and a subcellular localization of LIMK1 and LIMK2. The activity of LIMK2, distinct from LIMK1, toward cofilin phosphorylation did not change in the normal cell division cycle. In contrast, LIMK2 was hyperphosphorylated and its activity was markedly increased when HeLa cells were synchronized at mitosis with nocodazole treatment. Immunofluorescence analysis showed that LIMK1 was localized at cell-cell adhesion sites in interphase and prophase, redistributed to the spindle poles during prometaphase to anaphase, and accumulated at the cleavage furrow in telophase. In contrast, LIMK2 was diffusely localized in the cytoplasm during interphase, redistributed to the mitotic spindle, and finally to the spindle midzone during anaphase to telophase. These findings suggest that LIMK2 is activated in response to microtubule disruption, and that LIMK1 and LIMK2 may play different roles in regulating for the mitotic spindle organization, chromosome segregation, and cytokinesis during the cell division cycle.     

LIM kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning

The interaction of astral microtubules with cortical actin networks is essential for the correct orientation of the mitotic spindle; however, little is known about how the cortical actin organization is regulated during mitosis. LIM kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. LIMK1 activity increases during mitosis. Here we show that mitotic LIMK1 activation is critical for accurate spindle orientation in mammalian cells. Knockdown of LIMK1 suppressed a mitosis-specific increase in cofilin phosphorylation and caused unusual cofilin localization in the cell cortex in metaphase, instability of cortical actin organization and astral microtubules, irregular rotation and misorientation of the spindle, and a delay in anaphase onset. Similar results were obtained by treating the cells with a LIMK1 in hibitor peptide or latrunculin A or by overexpressing a non-phosphorylatable cofilin(S3A) mutant. Furthermore, localization of LGN (a protein containing the repetitive Leu-Gly-Asn tripeptide motifs), an important regulator of spindle orientation, in the crescent-shaped cortical regions was perturbed in LIMK1 knockdown cells. Our results suggest that LIMK1-mediated cofilin phosphorylation is required for accurate spindle orientation by stabilizing cortical actin networks during mitosis.     

Mitosis-dependent phosphorylation and activation of LIM-kinase 1.

LIM-kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylation of cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cell-cycle-dependent activity of endogenous LIMK1. When HeLa cells were synchronized at prometaphase by nocodazole-treatment, LIMK1 was hyperphosphorylated, and its activity toward cofilin phosphorylation was markedly increased. During cell cycle progression, LIMK1 activity was low in interphase but reached a maximal level during mitosis. Activation of LIMK1 during mitosis was abrogated by roscovitine, a specific inhibitor of cyclin-dependent kinases (CDKs), suggesting that activation of CDKs directly or indirectly participates in LIMK1 activation. These results strongly suggest that LIMK1 may play an important role in the cell cycle progression through regulation of actin cytoskeletal rearrangements.     

Cell cycle-associated changes in Slingshot phosphatase activity and roles in cytokinesis in animal cells

During cytokinesis the actomyosin-based contractile ring is formed at the equator, constricted, and then disassembled prior to cell abscission. Cofilin stimulates actin filament disassembly and is implicated in the regulation of contractile ring dynamics. However, little is known about the mechanism controlling cofilin activity during cytokinesis. Cofilin is inactivated by phosphorylation on Ser-3 by LIM-kinase-1 (LIMK1) and is reactivated by a protein phosphatase Slingshot-1 (SSH1). Here we show that the phosphatase activity of SSH1 decreases in the early stages of mitosis and is elevated in telophase and cytokinesis in HeLa cells, a time course correlating with that of cofilin dephosphorylation. SSH1 co-localizes with F-actin and accumulates onto the cleavage furrow and the midbody. Expression of a phosphatase-inactive SSH1 induces aberrant accumulation of F-actin and phospho-cofilin near the midbody in the final stage of cytokinesis and frequently leads to the regression of the cleavage furrow and the formation of multinucleate cells. Co-expression of cofilin rescued the inhibitory effect of phosphatase-inactive SSH1 on cytokinesis. These results suggest that SSH1 plays a critical role in cytokinesis by dephosphorylating and reactivating cofilin in later stages of mitosis.     

Accumulation of GTP-bound RhoA during cytokinesis and a critical role of ECT2 in this accumulation

We developed a new pull-down assay for GTP-Rho and examined its level during cell cycle. HeLa cells were arrested in the S phase by thymidine and were enriched in the prometaphase, metaphase, telophase, and G(1) phase by collecting at 0, 45, 90, and 180 min after the release from the nocodazole arrest, respectively. The level of GTP-Rho did not change significantly from the S phase to the prometaphase, but increased thereafter, peaking in the telophase, and returned to the original level in the G(1) phase. The GDP-GTP exchange activity for Rho measured in cell lysates in parallel increased also during the mitosis with a peak in the metaphase. Using this system, we examined a role of ECT2, an exchanger for Rho GTPases, suggested to be involved in cytokinesis (Tatsumoto, T., Xie, X., Blumenthal, R., Okamoto, I., and Miki., T. (1999) J. Cell. Biol. , 147, 921-928). Expression of the dominant negative form of ECT2 completely suppressed both the rise of GTP-Rho in the telophase and the increased GDP-GTP exchange activity in the mitotic cell extracts. These results suggest a critical role of ECT2 in Rho activation during cytokinesis.     

A temperature-sensitive CHO-K1 cell mutant (tsTM13) defective in chromosome decondensation and spindle deconstruction in M phase

A temperature-sensitive CHO-K1 cell mutant, tsTM13, exhibited a delayed cell cycle progression from metaphase to telophase at a nonpermissive temperature and was finally arrested from anaphase to telophase. Metaphase chromosomes were overcondensed and chromosome disjunction in anaphase was uncoordinated. In telophase, sister chromatids were segregated and cytokinesis was completed, but chromosome structure remained in a condensed state and the spindle was not deconstructed. The level of phosphorylation of histones H1 and H3 remained high in the later stages of mitosis and the activity of histone H1 kinase was also maintained at a high level. These results strongly suggest that the pleiotropic defects of tsTM13 cells in mitosis are associated with a lack of inactivation of activated histone H1 kinase.     

Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

Functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis more slowly after microtubule poisoning, than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be separable from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phospho-Bcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL(Ser49) is found with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit.     

Stromal Cell-Derived Factor 1α Activates LIM Kinase 1 and Induces Cofilin Phosphorylation for T-Cell Chemotaxis

Stromal cell-derived factor 1 alpha (SDF-1alpha), the ligand for G-protein-coupled receptor CXCR4, is a chemotactic factor for T lymphocytes. LIM kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing and -severing protein, at Ser-3 and regulates actin reorganization. We investigated the role of cofilin phosphorylation by LIMK1 in SDF-1alpha-induced chemotaxis of T lymphocytes. SDF-1alpha significantly induced the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1alpha also induced cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibited SDF-1alpha-induced LIMK1 activation, thus indicating that Gi protein is involved in LIMK1 activation. Expression of dominant negative Rac (DN-Rac), but not DN-Rho or DN-Cdc42, blocked SDF-1alpha-induced activation of LIMK1, which means that SDF-1alpha-induced LIMK1 activation is mediated by Rac but not by Rho or Cdc42. We used a cell-permeable peptide (S3 peptide) that contains the phosphorylation site (Ser-3) of cofilin to inhibit the cellular function of LIMK1. S3 peptide inhibited the kinase activity of LIMK1 in vitro. Treatment of Jurkat cells with S3 peptide inhibited the SDF-1alpha-induced cofilin phosphorylation, actin reorganization, and chemotactic response of Jurkat cells. These results suggest that the phosphorylation of cofilin by LIMK1 plays a critical role in the SDF-1alpha-induced chemotactic response of T lymphocytes.     

Myosin light chain kinases and phosphatase in mitosis and cytokinesis

At mitosis, cells undergo drastic alterations in morphology and cytoskeletal organization including cell rounding during prophase, mitotic spindle assembly during prometaphase and metaphase, chromatid segregation in anaphase, and cytokinesis during telophase. It is well established that myosin II is a motor responsible for cytokinesis. Recent reports have indicated that myosin II is also involved in spindle assembly and karyokinesis. In this review, we summarize current understanding of the functions of myosin II in mitosis and cytokinesis of higher eukaryotes, and discuss the roles of possible upstream molecules that control myosin II in these mitotic events.     

Par-3 mediates the inhibition of LIM kinase 2 to regulate cofilin phosphorylation and tight junction assembly

The polarity protein Par-3 plays critical roles in axon specification and the establishment of epithelial apico-basal polarity. Par-3 associates with Par-6 and atypical protein kinase C and is required for the proper assembly of tight junctions, but the molecular basis for its functions is poorly understood. We now report that depletion of Par-3 elevates the phosphorylated pool of cofilin, a key regulator of actin dynamics. Expression of a nonphosphorylatable mutant of cofilin partially rescues tight junction assembly in cells lacking Par-3, as does the depletion of LIM kinase 2 (LIMK2), an upstream kinase for cofilin. Par-3 binds to LIMK2 but not to the related kinase LIMK1. Par-3 inhibits LIMK2 activity in vitro, and overexpressed Par-3 suppresses cofilin phosphorylation that is induced by lysophosphatidic acid. Our findings identify LIMK2 as a novel target of Par-3 and uncover a molecular mechanism by which Par-3 could regulate actin dynamics during cell polarization.     

Nischarin inhibits LIM kinase to regulate cofilin phosphorylation and cell invasion

Nischarin is a novel protein that regulates cell migration by inhibiting p21-activated kinase (PAK). LIM kinase (LIMK) is a downstream effector of PAK, and it is known to play an important role in cell invasion. Here we show that nischarin also associates with LIMK to inhibit LIMK activation, cofilin phosphorylation, and LIMK-mediated invasion of breast cancer cells, suggesting that nischarin regulates cell invasion by negative modulation of the LIMK/cofilin pathway. The amino terminus of nischarin binds to the PDZ and kinase domains of LIMK. Although LIMK activation enhances the interaction with nischarin, only phosphorylation of threonine 508 of LIMK is crucial for the interaction. Inhibition of endogenous nischarin expression by RNA interference stimulates breast cancer cell invasion. Also, nischarin small interfering RNA (siRNA) enhances cofilin phosphorylation. In addition, knock-down of nischarin showed branched projection actin structures. Collectively these data indicate that nischarin siRNA may enhance random migration, resulting in stimulation of invasion.     

Activation of LIM kinases by myotonic dystrophy kinase-related Cdc42-binding kinase alpha

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through cofilin phosphorylation downstream of distinct Rho family GTPases. Pak1 and ROCK, respectively, activate LIMK1 and LIMK2 downstream of Rac and Rho; however, an effector protein kinase for LIMKs downstream of Cdc42 remains to be defined. We now report evidence that LIMK1 and LIMK2 activities toward cofilin phosphorylation are stimulated in cells by the co-expression of myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha), an effector protein kinase of Cdc42. In vitro, MRCKalpha phosphorylated the protein kinase domain of LIM kinases, and the site in LIMK2 phosphorylated by MRCKalpha proved to be threonine 505 within the activation segment. Expression of MRCKalpha induced phosphorylation of actin depolymerizing factor (ADF)/cofilin in cells, whereas MRCKalpha-induced ADF/cofilin phosphorylation was inhibited by the co-expression with the protein kinase-deficient form of LIM kinases. These results indicate that MRCKalpha phosphorylates and activates LIM kinases downstream of Cdc42, which in turn regulates the actin cytoskeletal reorganization through the phosphorylation and inactivation of ADF/cofilin.     

MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells.     

Aurora-A kinase maintains the fidelity of early and late mitotic events in HeLa cells

Aurora-A, a member of the Aurora/Ipl1-related kinase family, is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. Here, we have investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. We demonstrated that suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G2 cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When we injected anti-Aurora-A antibodies into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells.     

Localization of PKA phosphorylation site, Ser(2030), in the three-dimensional structure of cardiac ryanodine receptor

Cofilin regulates actin filament dynamics by stimulating actin filament disassembly and plays a critical role in cytokinesis and chemotactic migration. Aip1 (actin-interacting protein 1), also called WDR1 (WD-repeat protein 1), is a highly conserved WD-repeat protein in eukaryotes and promotes cofilin-mediated actin filament disassembly in vitro; however, little is known about the mechanisms by which Aip1 functions in cytokinesis and cell migration in mammalian cells. In the present study, we investigated the roles of Aip1 in cytokinesis and chemotactic migration of human cells by silencing the expression of Aip1 using siRNA (small interfering RNA). Knockdown of Aip1 in HeLa cells increased the percentage of multinucleate cells; this effect was reversed by expression of an active form of cofilin. In Aip1-knockdown cells, the cleavage furrow ingressed normally from anaphase to early telophase; however, an excessive accumulation of actin filaments was observed on the contractile ring in late telophase. These results suggest that Aip1 plays a crucial role in the completion of cytokinesis by promoting cofilin-mediated actin filament disassembly in telophase. We have also shown that Aip1 knockdown significantly suppressed chemokine-induced chemotactic migration of Jurkat T-lymphoma cells, and this was blocked by expression of an active form of cofilin. Whereas control cells mostly formed a single lamellipodium in response to chemokine stimulation, Aip1 knockdown cells abnormally exhibited multiple protrusions around the cells before and after cell stimulation. This indicates that Aip1 plays an important role in directional cell migration by restricting the stimulus-induced membrane protrusion to one direction via promoting cofilin activity.     

A PAK4-LIMK1 pathway drives prostate cancer cell migration downstream of HGF

Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Cell migration and invasion requires reorganisation of the actin cytoskeleton; processes mediated by the Rho family GTPases. p21 activated kinase 4 (PAK4), an effector of the Rho family protein Cdc42, is activated downstream of HGF. We report here the novel finding that in prostate cancer cells PAK4 binds to and phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner. We show for the first time that variations in the level of PAK4 expression change the level of cofilin phosphorylation in cells, a change we correlate with LIMK1 activity, cell morphology and migratory behaviour. We identify for the first time a direct and localised interaction between PAK4 and LIMK1 within cells using FRET: FLIM. Moreover we show here that HGF mediates this interaction which is concentrated in small foci at the cell periphery. PAK4 and LIMK1 act synergistically to increase cell migration speed, whilst a reduction in PAK4 expression decreases cell speed. It is well established that unphosphorylated (active) cofilin is a required to drive cell migration. Our results support a model whereby HGF-stimulated cell migration also requires a cofilin phosphorylation step that is mediated by PAK4.     

Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration

Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.     

LIM kinase 1 modulates opsonized zymosan-triggered activation of macrophage-like U937 cells. Possible involvement of phosphorylation of cofilin and reorganization of actin cytoskeleton.

We have previously reported that cofilin, an actin-binding protein, plays an important role in phagocyte functions, such as respiratory burst, phagocytosis, and chemotaxis. On the other hand, it was recently found that LIM motif-containing kinase (LIMK) phosphorylates cofilin. In this work, we investigated the roles of LIMK in activated phagocytes. The results of immunostaining showed that in dormant phagocytes the endogenous LIMK1 was diffusely distributed in the cytosol of macrophage-like U937 cells, and when activated by opsonized zymosan (OZ), it was translocated to plasma membranes. Green fluorescence protein (GFP)-conjugated LIMK was expressed in the phagocytes, and the GFP-positive cells were isolated by a fluorescence-activated cell sorter. The isolated wild-type LIMK-overexpressing cells produced superoxide at a rate that was 3.2-fold higher than that of only GFP-expressing control cells, whereas the respiratory burst of dominant negative LIMK1(D460A)-expressing cells decreased to 31% of that of the control cells. Phagocytic activity monitored by using Texas Red-labeled OZ was also decreased in the D460A-expressing cells. By immunoblotting using a specific anti-phosphorylated cofilin antibody, it was revealed that in the OZ-activated wild-type LIMK1-GFP-expressing cells, the phosphorylated cofilin increased by 2.3-fold, and that in the OZ-activated D460A-GFP-expressing cells, the phosphorylated cofilin decreased to 47% of that of only GFP-expressing cells (mock control). Furthermore, in the wild-type LIMK1-expressing cells, OZ-evoked increase in filamentous actin was markedly enhanced, whereas in the dominant negative LIMK1-expressing cells, the total level of F-actin was strongly suppressed. These results suggest that LIMK1 regulates the functions of phagocytes through phosphorylation of cofilin and enhances the formation of filamentous actin.     

Phosphorylation–dephosphorylation cycle of HP1α governs accurate mitotic progression

Heterochromatin protein 1α (HP1α), a bona fide factor of silent chromatin, is required for establishing as well as maintaining the higher-order chromatin structure in eukaryotes. HP1α is decorated with several post-translational modifications, and many of these are critical for its cellular functions. HP1α is heavily phosphorylated; however, its physiological relevance had remained to be completely understood. We have recently demonstrated that human HP1α is a mitotic target for NDR kinase, and the phosphorylation at the hinge region of HP1α at the G₂/M phase of the cell cycle is crucial for mitotic progression and Sgo1 loading at mitotic centromeres (Chakraborty et al., 2014). We now demonstrate that the dephosphorylation of HP1α within its hinge domain occurs during mitosis, specifically soon after prometaphase. In the absence of the hinge-specific HP1α phosphorylation, either as a consequence of depleting NDR1 or in cells expressing a non-phosphorylatable HP1α mutant, the cells arrest in prometaphase with several mitotic defects. In this study we show that NDR1-depleted cells expressing hinge-specific phosphomimetic HP1α mutant rescues the prometaphase arrest but displays defects in mitotic exit, suggesting that the dephosphorylation of HP1α is required for the completion of cytokinesis. Taken together, our results reveal that the phosphorylation–dephosphorylation cycle of HP1α orchestrates accurate progression of cells through mitosis.     

Effect of inhibition of the catalytic activity of cyclic AMP-dependent protein kinase on mitosis in PtK1 cells

Evidence has suggested that cyclic AMP, acting through activation of the type II cyclic AMP-dependent protein kinase, may play a role in the regulation of interphase and mitotic microtubules. In order to examine the potential role of the type II cAMP-dependent kinase during mitosis, dividing PtK1 cells were microinjected with two specific inhibitors of the catalytic activity of the type II kinase. These inhibitors were a specific protein inhibitor of cAMP-dependent protein kinase (PKI) and an affinity-purified polyclonal antiserum (anti-C) directed against the catalytic subunit of the kinase. Both have been shown previously to inhibit kinase activity in vitro. Microinjection of PKI during early- to mid-prophase significantly delayed the progression of the cells through mitosis, with the greatest delay occurring in metaphase. PKI injected during prometaphase also delayed progression through mitosis but to a lesser extent. Microinjection of anti-C during early- to mid-prophase also caused a significant delay in the completion of mitosis, with many cells becoming "hung up" in prometaphase. Anti-C injected during prometaphase had little effect on subsequent progression through mitosis. Microinjection of either anti-C or PKI during metaphase had no discernible effect. No effect on anaphase movement of chromosomes was observed with any treatment. These results provide further evidence that cAMP-dependent phosphorylation may be involved in the regulation of mitosis, although whether it acts directly through regulation of mitotic spindle microtubules is unclear.